Patent Application
20220154234
This disclosure relates to recombinant production of steviol glycosides, glycosides of steviol precursors, and steviol glycoside precursors in recombinant hosts. In particular, this disclosure relates to production of steviol glycosides comprising steviol-13-O-glucoside (13-SMG), steviol-19-O-glucoside (19-SMG), steviol-1,2-bioside, 1,2-stevioside, rubusoside, rebaudioside A (RebA), rebaudioside B (RebB), rebaudioside D (RebD), rebaudioside M (RebM), mono-glycosylated ent-kaurenoic acids, di-glycosylated ent-kaurenoic acids, tri-glycosylated ent-kaurenoic acids, tri-glycosylated ent-kaurenols, tri-glycosylated steviol glycosides, tetra-glycosylated steviol glycosides, penta-glycosylated steviol glycosides, hexa-glycosylated steviol glycosides, hepta-glycosylated steviol glycosides, or isomers thereof in recombinant hosts.
Sweeteners are well known as ingredients used most commonly in the food, beverage, or confectionary industries. The sweetener can either be incorporated into a final food product during production or for stand-alone use, when appropriately diluted, as a tabletop sweetener or an at-home replacement for sugars in baking. Sweeteners include natural sweeteners such as sucrose, high fructose corn syrup, molasses, maple syrup, and honey and artificial sweeteners such as aspartame, saccharine, and sucralose. Stevia extract is a natural sweetener that can be isolated and extracted from a perennial shrub, Stevia rebaudiana. Stevia is commonly grown in South America and Asia for commercial production of stevia extract. Stevia extract, purified to various degrees, is used commercially as a high intensity sweetener in foods and in blends or alone as a tabletop sweetener.
Chemical structures for several steviol glycosides are shown in
As recovery and purification of steviol glycosides from the Stevia plant have proven to be labor intensive and inefficient, there remains a need for a recombinant production system that can accumulate high yields of desired steviol glycosides, such as RebD and RebM. There also remains a need for improved production of steviol glycosides in recombinant hosts for commercial uses.
It is against the above background that the present invention provides certain advantages and advancements over the prior art.
Although this invention as disclosed herein is not limited to specific advantages or functionalities, the invention provides a recombinant host cell capable of producing one or more steviol glycosides and/or glycosylated steviol precursors, or a composition thereof, comprising:
wherein at least one of the genes is a recombinant gene.
In one aspect of the recombinant host cell disclosed herein:
In one aspect of the recombinant host cell disclosed herein: the UGT73C1 polypeptide comprises a polypeptide having at least 60% identity to an amino acid sequence set forth in SEQ ID NO:127, the UGT73C3 polypeptide comprises a polypeptide having at least 60% identity to an amino acid sequence set forth in SEQ ID NO:133, the UGT73C5 polypeptide comprises a polypeptide having at least 60% identity to an amino acid sequence set forth in SEQ ID NO:135, the UGT73C6 polypeptide comprises a polypeptide having at least 60% identity to an amino acid sequence set forth in SEQ ID NO:137, the UGT73E1 polypeptide comprises a polypeptide having at least 50% identity to an amino acid sequence set forth in SEQ ID NO:141, the UGT74D1 polypeptide comprises a polypeptide having at least 50% identity to an amino acid sequence set forth in SEQ ID NO:143, the UGT75B1 polypeptide comprises a polypeptide having at least 50% sequence identity to an amino acid sequence set forth in SEQ ID NO:145, the UGT75L6 polypeptide comprises a polypeptide having at least 60% sequence identity to an amino acid sequence set forth in SEQ ID NO:147, the UGT76E12 polypeptide comprises a polypeptide having at least 60% sequence identity to an amino acid sequence set forth in SEQ ID NO:153, the Olel polypeptide comprises a polypeptide having at least 55% identity to an amino acid sequence set forth in SEQ ID NO:177, the UGT5 polypeptide comprises a polypeptide having at least 65% identity to an amino acid sequence set forth in SEQ ID NO:181, the SA Gtase polypeptide comprises a polypeptide having at least 55% identity to an amino acid sequence set forth in SEQ ID NO:183, the UDPG1 polypeptide comprises a polypeptide having at least 50% sequence identity to an amino acid sequence set forth in SEQ ID NO:185, the UN1671 polypeptide comprises a polypeptide having at least 45% identity to an amino acid sequence set forth in SEQ ID NO:201, the UGT74F1 polypeptide comprises a polypeptide having at least 50% sequence identity to an amino acid sequence set forth in SEQ ID NO:203, the UGT75D1 polypeptide comprises a polypeptide having at least 50% sequence identity to an amino acid sequence set forth in SEQ ID NO:205, the UGT84B2 polypeptide comprises a polypeptide having at least 40% sequence identity to an amino acid sequence set forth in SEQ ID NO:207, the UGT74F2-like UGT polypeptide comprises a polypeptide having at least 55% identity to an amino acid sequence set forth in SEQ ID NO:211, the UGT73C7 polypeptide comprises a polypeptide having at least 60% identity to an amino acid sequence set forth in SEQ ID NO:139, the CaUGT3 polypeptide comprises a polypeptide having at least 50% identity to an amino acid sequence set forth in SEQ ID NO:169, the UN32491 polypeptide comprises a polypeptide having at least 50% identity to an amino acid sequence set forth in SEQ ID NO:199, and/or the CaUGT2 polypeptide comprises a polypeptide having at least 55% identity to an amino acid sequence set forth in SEQ ID NO:209.
In one aspect of the recombinant host cell disclosed herein, the recombinant host cell further comprises:
wherein at least one of the genes is a recombinant gene.
In one aspect of the recombinant host cell disclosed herein:
In one aspect of the recombinant host cell disclosed herein, expression of the one or more recombinant genes increases an amount of the one or more steviol glycosides and/or glycosylated steviol precursors, or a composition thereof accumulated by the cell relative to a corresponding host lacking the one or more recombinant genes.
In one aspect of the recombinant host cell disclosed herein, expression of the one or more recombinant genes increases the amount of the one or more steviol glycosides and/or glycosylated steviol precursors, or the composition thereof, accumulated by the cell by at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100% relative to a corresponding host lacking the one or more recombinant genes.
In one aspect of the recombinant host cell disclosed herein, expression of the one or more recombinant genes increases the amount of ent-kaurenoic acid+2Glc (#7), ent-kaurenoic acid+3Glc (isomer 1), ent-kaurenoic acid+3Glc (isomer 2), steviol-13-O-glucoside (13-SMG), Rebaudioside A (RebA), Rebaudioside B (RebB), Steviol+4Glc (#36), Steviol+6Glc (isomer 1), Steviol+7Glc (isomer 2), and/or ent-Kaurenol+3Glc (isomer 1 and/or isomer 2) accumulated by the cell relative to a corresponding host lacking the one or more recombinant genes.
In one aspect of the recombinant host cell disclosed herein, the one or more steviol glycosides and/or glycosylated steviol precursors are, or the composition thereof comprises, 13-SMG, steviol-19-O-glucoside (19-SMG), steviol-1,2-bioside, steviol-1,3-bioside, 1,2-stevioside, 1,3-stevioside, rubusoside, RebA, RebB, Rebaudioside C (RebC), Rebaudioside D (RebD), Rebaudioside E (RebE), Rebaudioside F (RebF), Rebaudioside M (RebM), Rebaudioside Q (RebQ), Rebaudioside I (RebI), dulcoside A, a mono-glycosylated ent-kaurenoic acid, a di-glycosylated ent-kaurenoic acid, a tri-glycosylated ent-kaurenoic acid, a mono-glycosylated ent-kaurenols, a di-glycosylated ent-kaurenol, a tri-glycosylated ent-kaurenol, a tri-glycosylated steviol glycoside, a tetra-glycosylated steviol glycoside, a penta-glycosylated steviol glycoside, a hexa-glycosylated steviol glycoside, a hepta-glycosylated steviol glycoside, or an isomer thereof.
In one aspect of the recombinant host cell disclosed herein, the mono-glycosylated ent-kaurenoic acid comprises KA1.58 of Table 1 and/or the penta-glycosylated steviol comprises Compound 5.24 of Table 1.
In one aspect of the recombinant host cell disclosed herein, the recombinant host cell comprises a plant cell, a mammalian cell, an insect cell, a fungal cell, an algal cell, or a bacterial cell.
The invention also provides a method of producing in a cell culture one or more steviol glycosides and/or glycosylated steviol precursors, or a composition thereof, comprising growing the recombinant host cell disclosed herein in the cell culture, under conditions in which the genes are expressed, and wherein the one or more steviol glycosides and/or glycosylated steviol precursors, or the composition thereof is produced by the recombinant host cell.
In one aspect of the method disclosed herein, the genes are constitutively expressed and/or expression of the genes is induced.
In one aspect of the method disclosed herein, an amount of ent-kaurenoic acid+2Glc (#7), ent-kaurenoic acid+3Glc (isomer 1), ent-kaurenoic acid+3Glc (isomer 2), 13-SMG, RebA, RebB, Steviol+4Glc (#36), Steviol+6Glc (isomer 1), Steviol+7Glc (isomer 2), and/or ent-Kaurenol+3Glc (isomer 1 and/or isomer 2) accumulated by the recombinant host cell is increased by at least about 5% relative to a corresponding host lacking the one or more recombinant genes.
In one aspect, the method disclosed herein further comprises isolating from the cell cultures the one or more steviol glycosides and/or glycosylated steviol precursors or the composition thereof produced thereby.
In one aspect of the method disclosed herein, the isolating step comprises:
In one aspect, the method disclosed herein further comprises recovering from the cell culture the one or more steviol glycosides and/or glycosylated steviol precursors or the composition thereof from the cell culture, wherein the cell culture is enriched for the one or more steviol glycosides and/or glycosides of a steviol presursor, or the composition thereof relative to a steviol glycoside composition from a Stevia plant and has a reduced level of Stevia plant-derived components relative to a plant-derived Stevia extract.
In one aspect of the method disclosed herein, the recovered one or more steviol glycosides and/or glycosylated steviol precursors, or the composition thereof are present in relative amounts that are different from a steviol glycoside composition recovered from a Stevia plant and have a reduced level of Stevia plant-derived components relative to a plant-derived Stevia extract.
The invention also provides a method for producing one or more steviol glycosides and/or glycosylated steviol precursors, or the composition thereof, comprising whole cell bioconversion of plant-derived or synthetic steviol, steviol precursors and/or steviol glycosides in a cell culture medium of a recombinant host using:
wherein at least one of the polypeptides is a recombinant polypeptide expressed in the recombinant host cell; and producing the one or more steviol glycosides and/or glycosylated steviol precursors, or the composition thereof, thereby.
In one aspect of the method disclosed herein:
In one aspect of the method disclosed herein, the UGT73C1 polypeptide comprises a polypeptide having at least 60% identity to an amino acid sequence set forth in SEQ ID NO:127, the UGT73C3 polypeptide comprises a polypeptide having at least 60% identity to an amino acid sequence set forth in SEQ ID NO:133, the UGT73C5 polypeptide comprises a polypeptide having at least 60% identity to an amino acid sequence set forth in SEQ ID NO:135, the UGT73C6 polypeptide comprises a polypeptide having at least 60% identity to an amino acid sequence set forth in SEQ ID NO:137, the UGT73E1 polypeptide comprises a polypeptide having at least 50% identity to an amino acid sequence set forth in SEQ ID NO:141, a UGT74D1 polypeptide comprises a polypeptide having at least 50% identity to an amino acid sequence set forth in SEQ ID NO:143, the UGT75B1 polypeptide comprises a polypeptide having at least 50% sequence identity to an amino acid sequence set forth in SEQ ID NO:145, the UGT75L6 polypeptide comprises a polypeptide having at least 60% sequence identity to an amino acid sequence set forth in SEQ ID NO:147, the UGT76E12 polypeptide comprises a polypeptide having at least 60% sequence identity to an amino acid sequence set forth in SEQ ID NO:153, the Olel polypeptide comprises a polypeptide having at least 55% identity to an amino acid sequence set forth in SEQ ID NO:177, the UGT5 polypeptide comprises a polypeptide having at least 65% identity to an amino acid sequence set forth in SEQ ID NO:181, the SA Gtase polypeptide comprises a polypeptide having at least 55% identity to an amino acid sequence set forth in SEQ ID NO:183, the UDPG1 polypeptide comprises a polypeptide having at least 50% sequence identity to an amino acid sequence set forth in SEQ ID NO:185, the UN1671 polypeptide comprises a polypeptide having at least 45% identity to an amino acid sequence set forth in SEQ ID NO:201, the UGT74F1 polypeptide comprises a polypeptide having at least 50% sequence identity to an amino acid sequence set forth in SEQ ID NO:203, the UGT75D1 polypeptide comprises a polypeptide having at least 50% sequence identity to an amino acid sequence set forth in SEQ ID NO:205, the UGT84B2 polypeptide comprises a polypeptide having at least 40% sequence identity to an amino acid sequence set forth in SEQ ID NO:207, the UGT74F2-like UGT polypeptide comprises a polypeptide having at least 55% identity to an amino acid sequence set forth in SEQ ID NO:211, the UGT73C7 polypeptide comprises a polypeptide having at least 60% identity to an amino acid sequence set forth in SEQ ID NO:139, the CaUGT3 polypeptide comprises a polypeptide having at least 50% identity to an amino acid sequence set forth in SEQ ID NO:169, the UN32491 polypeptide comprises a polypeptide having at least 50% identity to an amino acid sequence set forth in SEQ ID NO:199, or the CaUGT2 polypeptide comprises a polypeptide having at least 55% identity to an amino acid sequence set forth in SEQ ID NO:209.
In one aspect of the method disclosed herein, the recombinant host cell is a plant cell, a mammalian cell, an insect cell, a fungal cell, an algal cell or a bacterial cell.
The invention also provides an in vitro method for producing one or more steviol glycosides and/or glycosylated steviol precursors, or a composition thereof comprising adding:
and a plant-derived or synthetic steviol glycoside precursor or a plant-derived or synthetic steviol precursor to a reaction mixture;
wherein at least one of the polypeptides is a recombinant polypeptide; and
producing the one or more steviol glycosides and/or glycosylated steviol precursors, or the composition thereof, thereby.
In one aspect of the method disclosed herein, the reaction mixture comprises:
In one aspect of the method disclosed herein, the one or more steviol glycosides and/or glycosylated steviol precursors are, or the composition thereof comprises, 13-SMG, 19-SMG, steviol-1,2-bioside, steviol-1,3-bioside, 1,2-stevioside, 1,3-stevioside, rubusoside, RebA, RebB, RebC, RebD, RebE, RebF, RebM, RebQ, RebI, dulcoside A, a mono-glycosylated ent-kaurenoic acid, a di-glycosylated ent-kaurenoic acid, a tri-glycosylated ent-kaurenoic acid, a mono-glycosylated ent-kaurenols, a di-glycosylated ent-kaurenol, a tri-glycosylated ent-kaurenol, a tri-glycosylated steviol glycoside, a tetra-glycosylated steviol glycoside, a penta-glycosylated steviol glycoside, a hexa-glycosylated steviol glycoside, a hepta-glycosylated steviol glycoside, and/or an isomer thereof.
In one aspect of the method disclosed herein, the mono-glycosylated ent-kaurenoic acid comprises KA1.58 of Table 1 and/or the penta-glycosylated steviol comprises Compound 5.24 of Table 1.
The invention also provides a cell culture, comprising the recombinant host cell disclosed herein, the cell culture further comprising:
wherein the one or more steviol glycosides and/or glycosylated steviol precursors, or the composition thereof is present at a concentration of at least 1 mg/liter of the cell culture;
wherein the cell culture is enriched for the one or more steviol glycosides and/or glycosides of a steviol presursor, or the composition thereof relative to a steviol glycoside composition from a Stevia plant and has a reduced level of Stevia plant-derived components relative to a plant-derived Stevia extract.
The invention also provides a cell lysate from the recombinant host cell disclosed herein grown in the cell culture, comprising:
wherein the one or more steviol glycosides and/or glycosylated steviol precursors, or the composition thereof produced by the recombinant host cell is present at a concentration of at least 1 mg/liter of the cell culture.
The invention also provides a reaction mixture, comprising:
The invention also provides a composition of one or more steviol glycosides and/or glycosylated steviol precursors produced by the recombinant host cell disclosed herein; wherein the one or more steviol glycosides and/or glycosylated steviol precursors produced by the recombinant host cell are present in relative amounts that are different from a steviol glycoside composition from a Stevia plant and have a reduced level of Stevia plant-derived components relative to a plant-derived Stevia extract.
The invention also provides a composition of one or more steviol glycosides and/or glycosylated steviol precursors produced by the method disclosed herein; wherein the one or more steviol glycosides and/or glycosylated steviol precursors produced by the recombinant host cell are present in relative amounts that are different from a steviol glycoside composition from a Stevia plant and have a reduced level of Stevia plant-derived components relative to a plant-derived Stevia extract.
The invention also provides a sweetener composition, comprising one or more steviol glycosides and/or glycosylated steviol precursors produced by the recombinant host cell and/or the method disclosed herein.
The invention also provides a food product, comprising the sweetener composition disclosed herein.
The invention also provides a beverage or a beverage concentrate, comprising the sweetener composition disclosed herein.
The invention also provides an isolated nucleic acid molecule encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl position or a catalytically active portion thereof, wherein the encoded polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl position or the catalytically active portion thereof has at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:127, at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:133, at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:135, at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:137, at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:141, at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:145, at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:147, at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:177, at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO:181, at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:183, at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:185, at least 45% sequence identity to the amino acid sequence set forth in SEQ ID NO:201, at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:203, at least 40% sequence identity to the amino acid sequence set forth in SEQ ID NO:207, or at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:211.
The invention also provides an isolated nucleic acid molecule encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl position or a catalytically active portion thereof, wherein the encoded polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl position or the catalytically active portion thereof has at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:127, at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:133, at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:135, at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:137, at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:139, at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:141, or at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:153.
The invention also provides an isolated nucleic acid molecule encoding a polypeptide capable of beta-1,2-glycosylation of the C2′ and/or beta-1,3-glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside or a catalytically active portion thereof, wherein the encoded polypeptide capable of beta-1,2-glycosylation of the C2′ and/or beta-1,3-glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside or the catalytically active portion thereof has at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:137, at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:169, at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:199, or at least 45% sequence identity to the amino acid sequence set forth in SEQ ID NO:201.
The invention also provides an isolated nucleic acid molecule encoding a polypeptide capable of glycosylating a steviol precursor at its C-19 carboxyl or C-19 hydroxyl position or a catalytically active portion thereof, wherein the encoded polypeptide capable of glycosylating a steviol precursor at its C-19 carboxyl or C-19 hydroxyl position or the catalytically active portion thereof has at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:127, at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:133, at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:135, at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:137, at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:141, at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:145, at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:147, at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:153, at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:177, at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO:181, at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:183, at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:185, at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:203, at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:205, at least 40% sequence identity to the amino acid sequence set forth in SEQ ID NO:207, or at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:211.
In one aspect of the isolated nucleic acids disclosed herein, the nucleic acid is cDNA.
These and other features and advantages of the present invention will be more fully understood from the following detailed description taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description.
The following detailed description of the embodiments of the present invention can be best understood when read in conjunction with the following drawings, where like structure is indicated with like reference numerals and in which:
Skilled artisans will appreciate that elements in the Figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the dimensions of some of the elements in the Figures can be exaggerated relative to other elements to help improve understanding of the embodiment(s) of the present invention.
All publications, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes.
Before describing the present invention in detail, a number of terms will be defined. As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, reference to a “nucleic acid” means one or more nucleic acids.
It is noted that terms like “preferably,” “commonly,” and “typically” are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.
For the purposes of describing and defining the present invention it is noted that the term “substantially” is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term “substantially” is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.
Methods well known to those skilled in the art can be used to construct genetic expression constructs and recombinant cells according to this invention. These methods include in vitro recombinant DNA techniques, synthetic techniques, in vivo recombination techniques, and polymerase chain reaction (PCR) techniques. See, for example, techniques as described in Green & Sambrook, 2012, MOLECULAR CLONING: A LABORATORY MANUAL, Fourth Edition, Cold Spring Harbor Laboratory, New York; Ausubel et al., 1989, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, New York, and PCR Protocols: A Guide to Methods and Applications (Innis et al., 1990, Academic Press, San Diego, Calif.).
As used herein, the terms “polynucleotide,” “nucleotide,” “oligonucleotide,” and “nucleic acid” can be used interchangeably to refer to nucleic acid comprising DNA, RNA, derivatives thereof, or combinations thereof, in either single-stranded or double-stranded embodiments depending on context as understood by the skilled worker.
As used herein, the terms “microorganism,” “microorganism host,” and “microorganism host cell” can be used interchangeably. As used herein, the terms “recombinant host” and “recombinant host cell” can be used interchangeably. The person of ordinary skill in the art will appreciate that the terms “microorganism,” microorganism host,” and “microorganism host cell,” when used to describe a cell comprising a recombinant gene, may be taken to mean “recombinant host” or “recombinant host cell.” As used herein, the term “recombinant host” is intended to refer to a host, the genome of which has been augmented by at least one DNA sequence. Such DNA sequences include but are not limited to genes that are not naturally present, DNA sequences that are not normally transcribed into RNA or translated into a protein (“expressed”), and other genes or DNA sequences which one desires to introduce into a host. It will be appreciated that typically the genome of a recombinant host described herein is augmented through stable introduction of one or more recombinant genes. Generally, introduced DNA is not originally resident in the host that is the recipient of the DNA, but it is within the scope of this disclosure to isolate a DNA segment from a given host, and to subsequently introduce one or more additional copies of that DNA into the same host, e.g., to enhance production of the product of a gene or alter the expression pattern of a gene. In some instances, the introduced DNA will modify or even replace an endogenous gene or DNA sequence by, e.g., homologous recombination or site-directed mutagenesis. Suitable recombinant hosts include microorganisms.
As used herein, the term “recombinant gene” refers to a gene or DNA sequence that is introduced into a recipient host, regardless of whether the same or a similar gene or DNA sequence may already be present in such a host. “Introduced,” or “augmented” in this context, is known in the art to mean introduced or augmented by the hand of man. Thus, a recombinant gene can be a DNA sequence from another species or can be a DNA sequence that originated from or is present in the same species but has been incorporated into a host by recombinant methods to form a recombinant host. It will be appreciated that a recombinant gene that is introduced into a host can be identical to a DNA sequence that is normally present in the host being transformed, and is introduced to provide one or more additional copies of the DNA to thereby permit overexpression or modified expression of the gene product of that DNA. In some aspects, said recombinant genes are encoded by cDNA. In other embodiments, recombinant genes are synthetic and/or codon-optimized for expression in S. cerevisiae.
As used herein, the term “engineered biosynthetic pathway” refers to a biosynthetic pathway that occurs in a recombinant host, as described herein. In some aspects, one or more steps of the biosynthetic pathway do not naturally occur in an unmodified host. In some embodiments, a heterologous version of a gene is introduced into a host that comprises an endogenous version of the gene.
As used herein, the term “endogenous” gene refers to a gene that originates from and is produced or synthesized within a particular organism, tissue, or cell. In some embodiments, the endogenous gene is a yeast gene. In some embodiments, the gene is endogenous to S. cerevisiae, including, but not limited to S. cerevisiae strain S288C. In some embodiments, an endogenous yeast gene is overexpressed. As used herein, the term “overexpress” is used to refer to the expression of a gene in an organism at levels higher than the level of gene expression in a wild type organism. See, e.g., Prelich, 2012, Genetics 190:841-54. In some embodiments, an endogenous yeast gene, for example ADH, is deleted. See, e.g., Giaever & Nislow, 2014, Genetics 197(2):451-65. As used herein, the terms “deletion,” “deleted,” “knockout,” and “knocked out” can be used interchangabley to refer to an endogenous gene that has been manipulated to no longer be expressed in an organism, including, but not limited to, S. cerevisiae.
As used herein, the terms “heterologous sequence” and “heterologous coding sequence” are used to describe a sequence derived from a species other than the recombinant host. In some embodiments, the recombinant host is an S. cerevisiae cell, and a heterologous sequence is derived from an organism other than S. cerevisiae. A heterologous coding sequence, for example, can be from a prokaryotic microorganism, a eukaryotic microorganism, a plant, an animal, an insect, or a fungus different than the recombinant host expressing the heterologous sequence. In some embodiments, a coding sequence is a sequence that is native to the host.
A “selectable marker” can be one of any number of genes that complement host cell auxotrophy, provide antibiotic resistance, or result in a color change. Linearized DNA fragments of the gene replacement vector then are introduced into the cells using methods well known in the art (see below). Integration of the linear fragments into the genome and the disruption of the gene can be determined based on the selection marker and can be verified by, for example, PCR or Southern blot analysis. Subsequent to its use in selection, a selectable marker can be removed from the genome of the host cell by, e.g., Cre-LoxP systems (see, e.g., Gossen et al., 2002, Ann. Rev. Genetics 36:153-173 and U.S. 2006/0014264). Alternatively, a gene replacement vector can be constructed in such a way as to include a portion of the gene to be disrupted, where the portion is devoid of any endogenous gene promoter sequence and encodes none, or an inactive fragment of, the coding sequence of the gene.
As used herein, the terms “variant” and “mutant” are used to describe a protein sequence that has been modified at one or more amino acids, compared to the wild-type sequence of a particular protein.
As used herein, the term “inactive fragment” is a fragment of the gene that encodes a protein having, e.g., less than about 10% (e.g., less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or 0%) of the activity of the protein produced from the full-length coding sequence of the gene. Such a portion of a gene is inserted in a vector in such a way that no known promoter sequence is operably linked to the gene sequence, but that a stop codon and a transcription termination sequence are operably linked to the portion of the gene sequence. This vector can be subsequently linearized in the portion of the gene sequence and transformed into a cell. By way of single homologous recombination, this linearized vector is then integrated in the endogenous counterpart of the gene with inactivation thereof.
As used herein, the term “steviol glycoside” refers to rebaudioside A (RebA) (CAS #58543-16-1), rebaudioside B (RebB) (CAS #58543-17-2), rebaudioside C (RebC) (CAS #63550-99-2), rebaudioside D (RebD) (CAS #63279-13-0), rebaudioside E (RebE) (CAS #63279-14-1), rebaudioside F (RebF) (CAS #438045-89-7), rebaudioside M (RebM) (CAS #1220616-44-3), rubusoside (CAS #63849-39-4), Dulcoside A (CAS #64432-06-0), rebaudioside I (RebI) (MassBank Record: FU000332), rebaudioside Q (RebQ), 1,2-stevioside (CAS #57817-89-7), 1,3-stevioside (RebG), steviol-1,2-bioside (MassBank Record: FU000299), steviol-1,3-bioside, steviol-13-O-glucoside (13-SMG), steviol-19-O-glucoside (19-SMG), a tri-glucosylated steviol glycoside, a tetra-glycosylated steviol glycoside, a penta-glucosylated steviol glycoside, a hexa-glucosylated steviol glycoside, a hepta-glucosylated steviol glycoside, and isomers thereof. See
As used herein, the terms “steviol glycoside precursor” and “steviol glycoside precursor compound” are used to refer to intermediate compounds in the steviol glycoside biosynthetic pathway. Steviol glycoside precursors include, but are not limited to, geranylgeranyl diphosphate (GGPP), ent-copalyl-diphosphate, ent-kaurene, ent-kaurenol, ent-kaurenal, ent-kaurenoic acid, and steviol. See
As used herein, the term “contact” is used to refer to any physical interaction between two objects. For example, the term “contact” may refer to the interaction between an an enzyme and a substrate. In another example, the term “contact” may refer to the interaction between a liquid (e.g., a supernatant) and an adsorbent resin.
Steviol glycosides, steviol glycoside precursors, and/or glycosides of steviol precursors can be produced in vivo (i.e., in a recombinant host), in vitro (i.e., enzymatically), or by whole cell bioconversion. As used herein, the terms “produce” and “accumulate” can be used interchangeably to describe synthesis of steviol glycosides, glycosides of steviol precursors, and steviol glycoside precursors in vivo, in vitro, or by whole cell bioconversion.
Recombinant steviol glycoside-producing Saccharomyces cerevisiae (S. cerevisiae) strains are described in WO 2011/153378, WO 2013/022989, WO 2014/122227, and WO 2014/122328. Methods of producing steviol glycosides in recombinant hosts, by whole cell bio-conversion, and in vitro are also described in WO 2011/153378, WO 2013/022989, WO 2014/122227, and WO 2014/122328.
As used herein, the terms “culture broth,” “culture medium,” and “growth medium” can be used interchangeably to refer to a liquid or solid that supports growth of a cell. A culture broth can comprise glucose, fructose, sucrose, trace metals, vitamins, salts, yeast nitrogen base (YNB), and/or amino acids. The trace metals can be divalent cations, including, but not limited to, Mn2+ and/or Mg2+. In some embodiments, Mn2+ can be in the form of MnCl2 dihydrate and range from approximately 0.01 g/L to 100 g/L. In some embodiments, Mg2+ can be in the form of MgSO4 heptahydrate and range from approximately 0.01 g/L to 100 g/L. For example, a culture broth can comprise i) approximately 0.02-0.03 g/L MnCl2 dihydrate and approximately 0.5-3.8 g/L MgSO4 heptahydrate, ii) approximately 0.03-0.06 g/L MnCl2 dihydrate and approximately 0.5-3.8 g/L MgSO4 heptahydrate, and/or iii) approximately 0.03-0.17 g/L MnCl2 dihydrate and approximately 0.5-7.3 g/L MgSO4 heptahydrate. Additionally, a culture broth can comprise one or more steviol glycosides produced by a recombinant host, as described herein.
In some embodiments, a recombinant host comprising a gene encoding a polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP) (e.g., geranylgeranyl diphosphate synthase (GGPPS)); a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP (e.g., ent-copalyl diphosphate synthase (CDPS)); a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl diphosphate (e.g., kaurene synthase (KS)); a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenol from ent-kaurene (e.g., kaurene oxidase (KO)); a gene encoding a polypeptide capable of reducing cytochrome P450 complex (e.g., cytochrome P450 reductase (CPR) or P450 oxidoreductase (POR); for example, but not limited to a polypeptide capable of electron transfer from NADPH to cytochrome P450 complex during conversion of NADPH to NADP+, which is utilized as a cofactor for terpenoid biosynthesis); a gene encoding a polypeptide capable of synthesizing steviol from ent-kaurenoic acid (e.g., steviol synthase (KAH)); and/or a gene encoding a bifunctional polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP and synthesizing ent-kaurene from ent-copalyl diphosphate (e.g., an ent-copalyl diphosphate synthase (CDPS)—ent-kaurene synthase (KS) polypeptide) can produce steviol in vivo. See, e.g.,
In some embodiments, a recombinant host comprising a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl position (e.g., a UGT85C2 polypeptide); a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., a UGT76G1 polypeptide); a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl position (e.g., a UGT74G1 polypeptide); and/or a gene encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., a UGT91D2 or EUGT11 polypeptide) can produce a steviol glycoside in vivo. The skilled worker will appreciate that one or more of these genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host.
In some embodiments, steviol glycosides, glycosides of steviol precursors, and/or steviol glycoside precursors are produced in vivo through expression of one or more enzymes involved in the steviol glycoside biosynthetic pathway in a recombinant host. For example, a recombinant host comprising a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP; a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP; a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from ent-kaurene; a gene encoding a polypeptide capable of reducing cytochrome P450 complex; a gene encoding a bifunctional polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP and synthesizing ent-kaurene from ent-copalyl diphosphate; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl position; a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl position; and/or a gene encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside can produce a steviol glycoside and/or steviol glycoside precursors in vivo. See, e.g.,
In some aspects, the polypeptide capable of synthesizing GGPP from FPP and IPP comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:20 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:19), SEQ ID NO:22 (encoded by the nucleotide sequence set forth in SEQ ID NO:21), SEQ ID NO:24 (encoded by the nucleotide sequence set forth in SEQ ID NO:23), SEQ ID NO:26 (encoded by the nucleotide sequence set forth in SEQ ID NO:25), SEQ ID NO:28 (encoded by the nucleotide sequence set forth in SEQ ID NO:27), SEQ ID NO:30 (encoded by the nucleotide sequence set forth in SEQ ID NO:29), SEQ ID NO:32 (encoded by the nucleotide sequence set forth in SEQ ID NO:31), or SEQ ID NO:116 (encoded by the nucleotide sequence set forth in SEQ ID NO:115).
In some aspects, the polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:34 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:33), SEQ ID NO:36 (encoded by the nucleotide sequence set forth in SEQ ID NO:35), SEQ ID NO:38 (encoded by the nucleotide sequence set forth in SEQ ID NO:37), SEQ ID NO:40 (encoded by the nucleotide sequence set forth in SEQ ID NO:39), or SEQ ID NO:42 (encoded by the nucleotide sequence set forth in SEQ ID NO:41). In some embodiments, the polypeptide capable of synthesizing ent-copalyldiphosphate from GGPP lacks a chloroplast transit peptide.
In some aspects, the polypeptide capable of synthesizing ent-kaurene from ent-copalyl pyrophosphate comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:44 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:43), SEQ ID NO:46 (encoded by the nucleotide sequence set forth in SEQ ID NO:45), SEQ ID NO:48 (encoded by the nucleotide sequence set forth in SEQ ID NO:47), SEQ ID NO:50 (encoded by the nucleotide sequence set forth in SEQ ID NO:49), or SEQ ID NO:52 (encoded by the nucleotide sequence set forth in SEQ ID NO:51).
In some embodiments, a recombinant host comprises a gene encoding a bifunctional polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP and synthesizing ent-kaurene from ent-copalyl pyrophosphate. In some aspects, the bifunctional polypeptide comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:54 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:53), SEQ ID NO:56 (encoded by the nucleotide sequence set forth in SEQ ID NO:55), or SEQ ID NO:58 (encoded by the nucleotide sequence set forth in SEQ ID NO:57).
In some aspects, the polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenol from ent-kaurene comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:60 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:59), SEQ ID NO:62 (encoded by the nucleotide sequence set forth in SEQ ID NO:61), SEQ ID NO:117 (encoded by the nucleotide sequence set forth in SEQ ID NO:63 or SEQ ID NO:64), SEQ ID NO:66 (encoded by the nucleotide sequence set forth in SEQ ID NO:65), SEQ ID NO:68 (encoded by the nucleotide sequence set forth in SEQ ID NO:67), SEQ ID NO:70 (encoded by the nucleotide sequence set forth in SEQ ID NO:69), SEQ ID NO:72 (encoded by the nucleotide sequence set forth in SEQ ID NO:71), SEQ ID NO:74 (encoded by the nucleotide sequence set forth in SEQ ID NO:73), or SEQ ID NO:76 (encoded by the nucleotide sequence set forth in SEQ ID NO:75).
In some aspects, the polypeptide capable of reducing cytochrome P450 complex comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:78 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:77), SEQ ID NO:80 (encoded by the nucleotide sequence set forth in SEQ ID NO:79), SEQ ID NO:82 (encoded by the nucleotide sequence set forth in SEQ ID NO:81), SEQ ID NO:84 (encoded by the nucleotide sequence set forth in SEQ ID NO:83), SEQ ID NO:86 (encoded by the nucleotide sequence set forth in SEQ ID NO:85), SEQ ID NO:88 (encoded by the nucleotide sequence set forth in SEQ ID NO:87), SEQ ID NO:90 (encoded by the nucleotide sequence set forth in SEQ ID NO:89), or SEQ ID NO:92 (encoded by the nucleotide sequence set forth in SEQ ID NO:91).
In some aspects, the polypeptide capable of synthesizing steviol from ent-kaurenoic acid comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:94 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:93), SEQ ID NO:97 (encoded by the nucleotide sequence set forth in SEQ ID NO:95 or SEQ ID NO:96), SEQ ID NO:100 (encoded by the nucleotide sequence set forth in SEQ ID NO:98 or SEQ ID NO:99), SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:106 (encoded by the nucleotide sequence set forth in SEQ ID NO:105), SEQ ID NO:108 (encoded by the nucleotide sequence set forth in SEQ ID NO:107), SEQ ID NO:110 (encoded by the nucleotide sequence set forth in SEQ ID NO:109), SEQ ID NO:112 (encoded by the nucleotide sequence set forth in SEQ ID NO:111), or SEQ ID NO:114 (encoded by the nucleotide sequence set forth in SEQ ID NO:113).
In some embodiments, a recombinant host comprises a nucleic acid encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl position, a nucleic acid encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, a nucleic acid encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl position, a nucleic acid encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside. In certain such embodiments, the recombinant host further comprises a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP; a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP; a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from ent-kaurene; a gene encoding a polypeptide capable of reducing cytochrome P450 complex; and/or a gene encoding a bifunctional polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP and synthesizing ent-kaurene from ent-copalyl diphosphate.
In some embodiments, a recombinant host comprises a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl position, e.g., a UGT73C1 polypeptide, a UGT73C3 polypeptide, a UGT73C5 polypeptide, a UGT73C6 polypeptide, a UGT73E1 polypeptide, a UGT75B1 polypeptide, a UGT75L6 polypeptide, a Olel polypeptide, a UGT5 polypeptide, a SA Gtase polypeptide, a UDPG1 polypeptide, a UN1671 polypeptide, a UGT74F1 polypeptide, a UGT84B2 polypeptide, and/or a UGT74F2-like UGT polypeptide. In certain such embodiments, the recombinant host further comprises a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP; a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP; a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from ent-kaurene; a gene encoding a polypeptide capable of reducing cytochrome P450 complex; a gene encoding a bifunctional polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP and synthesizing ent-kaurene from ent-copalyl diphosphate; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl position; a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; and/or a gene encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside.
In some embodiments, a recombinant host comprises a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl position, e.g., a UGT73C1 polypeptide, a UGT73C3 polypeptide, a UGT73C5 polypeptide, a UGT73C6 polypeptide, a UGT73C7 polypeptide, a UGT73E1 polypeptide, and/or a UGT76E12 polypeptide. In certain such embodiments, the recombinant host further comprises a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP; a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP; a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from ent-kaurene; a gene encoding a polypeptide capable of reducing cytochrome P450 complex; a gene encoding a bifunctional polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP and synthesizing ent-kaurene from ent-copalyl diphosphate; a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl position; and/or a gene encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside.
In some embodiments, a recombinant host comprises a gene encoding a polypeptide capable of beta-1,2-glycosylation of the C2′ and/or beta-1,3-glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (that is, examples of glycosyl-position glycosylation), e.g., a UGT73C6 polypeptide, a CaUGT3 polypeptide, a UN32491 polypeptide, and/or a UN1671 polypeptide. In certain such embodiments, the recombinant host further comprises a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP; a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP; a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from ent-kaurene; a gene encoding a polypeptide capable of reducing cytochrome P450 complex; a gene encoding a bifunctional polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP and synthesizing ent-kaurene from ent-copalyl diphosphate; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl position; a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl position; and/or a gene encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside.
In some embodiments, a recombinant host comprises a gene encoding a polypeptide capable of glycosylating a steviol precursor at its C-19 carboxyl or C-19 hydroxyl position, e.g., a UGT73C1 polypeptide, a UGT73C3 polypeptide, a UGT73C5 polypeptide, a UGT73C6 polypeptide, a UGT73E1 polypeptide, a UGT75B1 polypeptide, a UGT75L6 polypeptide, a UGT76E12 polypeptide, a Olel polypeptide, a UGT5 polypeptide, a SA Gtase, a UDPG1 polypeptide, a UGT74F1 polypeptide, a UGT75D1 polypeptide, a UGT84B2 polypeptide, and/or a UGT74F2-like UGT polypeptide. In certain such embodiments, the recombinant host further comprises a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP; a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP; a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from ent-kaurene; a gene encoding a polypeptide capable of reducing cytochrome P450 complex; a gene encoding a bifunctional polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP and synthesizing ent-kaurene from ent-copalyl diphosphate; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl position; a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl position; and/or a gene encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside.
In some embodiments, a recombinant host comprises a nucleic acid encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl position (e.g., UGT85C2 polypeptide) (SEQ ID NO:7), a nucleic acid encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., UGT76G1 polypeptide) (SEQ ID NO:9), a nucleic acid encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl position (e.g., UGT74G1 polypeptide) (SEQ ID NO:4), a nucleic acid encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., EUGT11 polypeptide) (SEQ ID NO:16). In some aspects, the polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-0-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., UGT91D2 polypeptide) can be a UGT91D2e polypeptide (SEQ ID NO:11) or a UGT91D2e-b polypeptide (SEQ ID NO:13).
In some aspects, the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl position is encoded by the nucleotide sequence set forth in SEQ ID NO:5 or SEQ ID NO:6, the polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-0-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside is encoded by the nucleotide sequence set forth in SEQ ID NO:8, the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl position is encoded by the nucleotide sequence set forth in SEQ ID NO:3, the polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside is encoded by the nucleotide sequence set forth in SEQ ID NO:10,12,14 or 15. The skilled worker will appreciate that expression of these genes may be necessary to produce a particular steviol glycoside but that one or more of these genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host.
In a particular embodiment, a steviol-producing recombinant microorganism comprises exogenous nucleic acids encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl position, a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, and a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside polypeptides.
In another particular embodiment, a steviol-producing recombinant microorganism comprises exogenous nucleic acids encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl position; a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl position; and a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside.
In some embodiments, polypeptides capable of catalyzing the 19-O-glycosylation of ent-kaurenoic acid (KA) to ent-kaurenoic acid+1Glc (#58), in vitro, in a recombinant host, or by whole cell bioconversion include UGT73C1 (SEQ ID NO:127), UGT73C3 (SEQ ID NO:133), UGT73C5 (SEQ ID NO:135), UGT73C6 (SEQ ID NO:137), UGT73E1 (SEQ ID NO:141), UGT74G1 (SEQ ID NO:4), UGT75B1 (SEQ ID NO:145), UGT75L6 (SEQ ID NO:147), UGT76E12 (SEQ ID NO:153), Olel (SEQ ID NO:177), UGT5 (SEQ ID NO:181), SA Gtase (SEQ ID NO:183), UDPG1 (SEQ ID NO:185), UGT74F1 (SEQ ID NO:203), UGT75D1 (SEQ ID NO:205), UGT84B2 (SEQ ID NO:207), CaUGT2 (SEQ ID NO:209), and a UGT74F2-like UGT polypeptide (SEQ ID NO:211). See, Example 3.
In some embodiments, polypeptides capable of catalyzing the 13-O-glycosylation of steviol to 13-SMG, in vitro, in a recombinant host, or by whole cell bioconversion include UGT73C1 (SEQ ID NO:127), UGT73C3 (SEQ ID NO:133), UGT73C5 (SEQ ID NO:135), UGT73C6 (SEQ ID NO:137), UGT73C7 (SEQ ID NO:139), UGT73E1 (SEQ ID NO:141), UGT76E12 (SEQ ID NO:153), and UGT85C2 (SEQ ID NO:7). See, Example 3.
In some embodiments, polypeptides capable of catalyzing the 19-O-glycosylation of steviol to 19-SMG, in vitro, in a recombinant host, or by whole cell bioconversion include UGT73C1 (SEQ ID NO:127), UGT73C3 (SEQ ID NO:133), UGT73C5 (SEQ ID NO:135), UGT73C6 (SEQ ID NO:137), UGT73E1 (SEQ ID NO:141), UGT74D1 (SEQ ID NO:143), UGT74G1 (SEQ ID NO:4), UGT75B1 (SEQ ID NO:145), UGT75L6 (SEQ ID NO:147), Olel (SEQ ID NO:177), UGT5 (SEQ ID NO:181), SA Gtase (SEQ ID NO:183), and UDPG1 (SEQ ID NO:185). See, Example 3.
In some embodiments, polypeptides capable of catalyzing the 19-O-glycosylation of 13-SMG to rubusoside, in vitro, in a recombinant host, or by whole cell bioconversion include UGT73C1 (SEQ ID NO:127), UGT73C6 (SEQ ID NO:137), UGT74G1 (SEQ ID NO:4), UGT85C2 (SEQ ID NO:7), SA Gtase (SEQ ID NO:183), UDPG1 (SEQ ID NO:185), UN1671 (SEQ ID NO:201), UGT74F1 (SEQ ID NO:203), UGT75D1 (SEQ ID NO:205), UGT84B2 (SEQ ID NO:207), CaUGT2 (SEQ ID NO:209), and a UGT74F2-like UGT polypeptide (SEQ ID NO:211). See, Example 3.
In some embodiments, polypeptides capable of catalyzing the glycosylation of 13-SMG (that is, an examples of glycosyl-position glycosylation) to steviol-1,2-bioside, in vitro, in a recombinant host, or by whole cell bioconversion include UGT91D2e-b (SEQ ID NO:13), EUGT11 (SEQ ID NO:16), and UN32491 (SEQ ID NO:199).
In some embodiments, polypeptides capable of catalyzing the glycosyl-position glycosylation of rubusoside to 1,2-stevioside, in vitro, in a recombinant host, or by whole cell bioconversion include UGT73C6 (SEQ ID NO:137), UGT91D2e-b (SEQ ID NO:13), CaUGT3 (SEQ ID NO:169), and EUGT11 (SEQ ID NO:16). See, Example 3.
In some embodiments, polypeptides capable of catalyzing the glycosyl-position glycosylation of rubusoside to steviol+3Glc (#55), in vitro, in a recombinant host, or by whole cell bioconversion include EUGT11 (SEQ ID NO:16).
In some embodiments, polypeptides capable of catalyzing the 19-O-glycosylation of RebB to RebA, in vitro, in a recombinant host, or by whole cell bioconversion include UGT74G1 (SEQ ID NO:4). See, Example 3.
In some embodiments, polypeptides capable of catalyzing the glycosyl-position glycosylation of RebA to RebD, in vitro, in a recombinant host, or by whole cell bioconversion include EUGT11 (SEQ ID NO:16).
In some embodiments, polypeptides capable of catalyzing the glycosyl-position glycosylation of RebA to steviol+5Glc (#24), in vitro, in a recombinant host, or by whole cell bioconversion include EUGT11 (SEQ ID NO:16) and UN1671 (SEQ ID NO:201). See, Example 3.
In some aspects, polypeptides capable of 19-O-glycosylation activity on steviol, steviol glycosides, and precurors thereof in vitro, in a recombinant host, or by whole cell bioconversion include UGT73C1 (SEQ ID NO:127), UGT73C3 (SEQ ID NO:133), UGT73C5 (SEQ ID NO:135), UGT73C6 (SEQ ID NO:137), UGT73E1 (SEQ ID NO:141), UGT74G1 (SEQ ID NO:4), UGT85C2 (SEQ ID NO:7), UGT75B1 (SEQ ID NO:145), UGT75L6 (SEQ ID NO:147), UGT76E12 (SEQ ID NO:153), Olel (SEQ ID NO:177), UGT5 (SEQ ID NO:181), SA Gtase (SEQ ID NO:183), UDPG1 (SEQ ID NO:185), UN1671 (SEQ ID NO:201), UGT74F1 (SEQ ID NO:203), UGT75D1 (SEQ ID NO:205), UGT84B2 (SEQ ID NO:207), and a UGT74F2-like UGT (SEQ ID NO:211). See, Example 3. Non-limiting examples of 19-O-glycosylation reactions include conversion of ent-kaurenoic acid to ent-kaurenoic acid+1Glc (#58), conversion of 13-SMG to rubusoside, and/or conversion of steviol to 19-SMG (see, e.g.,
In some aspects, polypeptides capable of 13-O-glycosylation activity on steviol and steviol glycosides in vitro, in a recombinant host, or by whole cell bioconversion include UGT73C1 (SEQ ID NO:127), UGT73C3 (SEQ ID NO:133), UGT73C5 (SEQ ID NO:135), UGT73C6 (SEQ ID NO:137), UGT73C7 (SEQ ID NO:139), UGT73E1 (SEQ ID NO:141), UGT76E12 (SEQ ID NO:153), and UGT85C2 (SEQ ID NO:7). See, Example 3. A non-limiting example of a 13-O-glycosylation reaction includes conversion of steviol to 13-SMG (see, e.g.,
In some aspects, polypeptides capable of glycosylation activity towards the glucose residues of steviol glycosides including, but not limited to, catalyzing the conversion of 13-SMG to steviol-1,2-bioside, catalyzing the conversion of rubusoside to 1,2-stevioside, and/or catalyzing the conversion of RebA to steviol+5Glc (#24) (see, e.g.,
In some embodiments, a recombinant host comprises a nucleic acid encoding a UGT85C2 polypeptide (SEQ ID NO:7), a nucleic acid encoding a UGT76G1 polypeptide (SEQ ID NO:9), a nucleic acid encoding a UGT74G1 polypeptide (SEQ ID NO:4), a nucleic acid encoding a UGT91D2 polypeptide, and/or a nucleic acid encoding a EUGT11 polypeptide (SEQ ID NO:16). In some aspects, the UGT91D2 polypeptide can be a UGT91D2e polypeptide (SEQ ID NO:11) a UGT91D2e-b polypeptide (SEQ ID NO:13). In some embodiments, a recombinant host comprises a nucleic acid encoding a UGT73C1 polypeptide (SEQ ID NO:127), a nucleic acid encoding a UGT73C3 polypeptide (SEQ ID NO:133), a nucleic acid encoding a UGT73C5 polypeptide (SEQ ID NO:135), a nucleic acid encoding a UGT73C6 polypeptide (SEQ ID NO:137), a nucleic acid encoding a UGT73C7 polypeptide (SEQ ID NO:139), a nucleic acid encoding a UGT73E1 polypeptide (SEQ ID NO:141), a nucleic acid encoding a UGT74D1 polypeptide (SEQ ID NO:143), a nucleic acid encoding a UGT75B1 polypeptide (SEQ ID NO:145), a nucleic acid encoding a UGT75L6 polypeptide (SEQ ID NO:147), a nucleic acid encoding a UGT76E12 polypeptide (SEQ ID NO:153), a nucleic acid encoding a CaUGT3 polypeptide (SEQ ID NO:169), a nucleic acid encoding a Olel polypeptide (SEQ ID NO:177), a nucleic acid encoding a UGT5 (SEQ ID NO:181), a nucleic acid encoding a SA Gtase polypeptide (SEQ ID NO:183), a nucleic acid encoding a UDPG1 polypeptide (SEQ ID NO:185), a nucleic acid encoding a UN32491 polypeptide (SEQ ID NO:199), a nucleic acid encoding a UN1671 polypeptide (SEQ ID NO:201), a nucleic acid encoding a UGT74F1 polypeptide (SEQ ID NO:203), a nucleic acid encoding a UGT75D1 polypeptide (SEQ ID NO:205), a nucleic acid encoding a UGT84B2 polypeptide (SEQ ID NO:207), a nucleic acid encoding a CaUGT2 polypeptide (SEQ ID NO:209) or a nucleic acid encoding a UGT74F2-like UGT polypeptide (SEQ ID NO:211).
In some aspects, the UGT85C2 polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:5, SEQ ID NO:6 the UGT76G1 polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:8, the UGT74G1 polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:3 or SEQ ID NO:213, the UGT91D2e polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:10, the UGT91D2e-b polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:12 or SEQ ID NO:212, the EUGT11 polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:14 or SEQ ID NO:15, the UGT73C1 polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:126, the UGT73C3 polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:132, the UGT73C5 polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:134, the UGT73C6 polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:136, the UGT73C7 polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:138, the UGT73E1 polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:140, the UGT74D1 polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:142, the UGT75B1 polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:144, the UGT75L6 polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:146, the UGT76E12 polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:152, the CaUGT3 polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:168, the Olel polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:176, the UGT5 polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:180, the SA Gtase polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:182, the UDPG1 polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:184, the UN32491 polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:198, the UN1671 polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:200, the UGT74F1 polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:202, the UGT75D1 polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:204, the UGT84B2 polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:206, the CaUGT2 polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:208, and the UGT74F2-like UGT polypeptide is encoded by the nucleotide sequence set forth in SEQ ID NO:210.
In some embodiments, steviol glycosides, glycosides of steviol precursors, and/or steviol glycoside precursors are produced through contact of a steviol glycoside precursor with one or more enzymes involved in the steviol glycoside pathway in vitro. For example, contacting steviol with one or more of a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, and a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl position or a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl position can result in production of a steviol glycoside in vitro. In some embodiments, a steviol glycoside precursor is produced through contact of an upstream steviol glycoside precursor with one or more enzymes involved in the steviol glycoside pathway in vitro. For example, contacting ent-kaurenoic acid with a polypeptide capable of synthesizing steviol from ent-kaurenoic acid can result in production of steviol in vitro.
In some embodiments, one or more steviol glycosides and/or glycosylated steviol precursors, or a composition thereof are produced in vitro. In some embodiments the method comprises adding a UGT85C2 polypeptide having at least 55% identity to an amino acid sequence set forth in SEQ ID NO:7; a UGT76G1 polypeptide having at least 50% identity to an amino acid sequence set forth in SEQ ID NO:9; a UGT74G1 polypeptide having at least 55% identity to an amino acid sequence set forth in SEQ ID NO:4; a UGT91D2 functional homolog polypeptide comprising a UGT91D2e polypeptide having 90% or greater identity to an amino acid sequence set forth in SEQ ID NO:11 or a UGT91D2e-b polypeptide having 90% or greater identity to an amino acid sequence set forth in SEQ ID NO:13; a EUGT11 polypeptide having at least 65% identity to an amino acid sequence set forth in SEQ ID NO:16; a UGT73C1 polypeptide comprises a polypeptide having at least 60% identity to an amino acid sequence set forth in SEQ ID NO:127; a UGT73C3 polypeptide comprises a polypeptide having at least 60% identity to an amino acid sequence set forth in SEQ ID NO:133; a UGT73C5 polypeptide comprises a polypeptide having at least 60% identity to an amino acid sequence set forth in SEQ ID NO:135; a UGT73C6 polypeptide comprises a polypeptide having at least 60% identity to an amino acid sequence set forth in SEQ ID NO:137; a UGT73E1 polypeptide comprises a polypeptide having at least 50% identity to an amino acid sequence set forth in SEQ ID NO:141; a UGT75B1 polypeptide comprises a polypeptide having at least 50% sequence identity to an amino acid sequence set forth in SEQ ID NO:145; a UGT75L6 polypeptide comprises a polypeptide having at least 60% sequence identity to an amino acid sequence set forth in SEQ ID NO:147; a UGT76E12 polypeptide comprises a polypeptide having at least 60% sequence identity to an amino acid sequence set forth in SEQ ID NO:153; a Olel polypeptide comprises a polypeptide having at least 55% identity to an amino acid sequence set forth in SEQ ID NO:177; a UGT5 polypeptide comprises a polypeptide having at least 65% identity to an amino acid sequence set forth in SEQ ID NO:181; a SA Gtase polypeptide comprises a polypeptide having at least 55% identity to an amino acid sequence set forth in SEQ ID NO:183; a UDPG1 polypeptide comprises a polypeptide having at least 50% sequence identity to an amino acid sequence set forth in SEQ ID NO:185; a UN1671 polypeptide comprises a polypeptide having at least 45% identity to an amino acid sequence set forth in SEQ ID NO:201; a UGT74F1 polypeptide comprises a polypeptide having at least 50% sequence identity to an amino acid sequence set forth in SEQ ID NO:203; a UGT75D1 polypeptide comprises a polypeptide having at least 50% sequence identity to an amino acid sequence set forth in SEQ ID NO:205; a UGT84B2 polypeptide comprises a polypeptide having at least 40% sequence identity to an amino acid sequence set forth in SEQ ID NO:207; a UGT74F2-like UGT polypeptide comprises a polypeptide having at least 55% identity to an amino acid sequence set forth in SEQ ID NO:211; a UGT73C7 polypeptide comprises a polypeptide having at least 60% identity to an amino acid sequence set forth in SEQ ID NO:139; a CaUGT3 polypeptide comprises a polypeptide having at least 50% identity to an amino acid sequence set forth in SEQ ID NO:169; and/or a UN32491 polypeptide comprises a polypeptide having at least 50% identity to an amino acid sequence set forth in SEQ ID NO:199; and a plant-derived or synthetic steviol glycoside precursor or a plant-derived or synthetic steviol to a reaction mixture; wherein at least one of the polypeptides is a recombinant polypeptide; and producing the one or more steviol glycosides and/or glycosylated steviol precursors, or the composition thereof, thereby.
In some embodiments, a steviol glycoside or steviol glycoside precursor is produced by whole cell bioconversion. For whole cell bioconversion to occur, a host cell expressing one or more enzymes involved in the steviol glycoside pathway takes up and modifies the steviol glycoside or steviol glycoside precursor in the cell; following modification in vivo, the steviol glycoside or steviol glycoside precursor remains in the cell and/or is excreted into the cell culture medium. For example, a host cell expressing a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl position; a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl position; and/or a gene encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside can take up steviol and glycosylate steviol in the cell; following glycosylation in vivo, a steviol glycoside can be excreted into the culture medium. In certain such embodiments, the host cell may further express a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP; a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP; a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from ent-kaurene; a gene encoding a polypeptide capable of reducing cytochrome P450 complex; a gene encoding a polypeptide capable of synthesizing steviol from ent-kaurenoic acid; and/or a gene encoding a bifunctional polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP and synthesizing ent-kaurene from ent-copalyl diphosphate.
In some embodiments, the method for producing one or more steviol glycosides and/or glycosylated steviol precursors, or a composition thereof as disclosed herein comprises whole cell bioconversion of a plant-derived or synthetic steviol glycoside precursor or a plant-derived or synthetic steviol precursor in a cell culture medium of a recombinant host cell using (a) a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl position; (b) a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl position; (c) a polypeptide capable of beta-1,2-glycosylation of the C2′ and/or beta-1,3-glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (that is, examples of glycosyl-position glycosylation) activity on a steviol glycoside; and/or (d) a polypeptide is capable of glycosylating a steviol precursor at its C-19 carboxyl or C-19 hydroxyl position; wherein at least one of the polypeptide is a recombinant polypeptide expressed in the recombinant host cell, and producing the one or more steviol glycosides and/or glycosylated steviol precursors, or a composition thereof, thereby.
In some embodiments of the method for producing one or more steviol glycosides and/or glycosylated steviol precursors, or a composition thereof as disclosed herein by whole cell bioconversion of a plant-derived or synthetic steviol glycoside precursor or a plant-derived or synthetic steviol precursor in a cell culture medium of a recombinant host cell described herein, the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl position comprises a UGT73C1 polypeptide, a UGT73C3 polypeptide, a UGT73C5 polypeptide, a UGT73C6 polypeptide, a UGT73E1 polypeptide, a UGT75B1 polypeptide, a UGT75L6 polypeptide, a Olel polypeptide, a UGT5 polypeptide, a SA Gtase polypeptide, a UDPG1 polypeptide, a UN1671 polypeptide, a UGT74F1 polypeptide, a UGT84B2 polypeptide, and/or a UGT74F2-like UGT polypeptide; the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl position comprises a UGT73C1 polypeptide, a UGT73C3 polypeptide, a UGT73C5 polypeptide, a UGT73C6 polypeptide, a UGT73C7 polypeptide, a UGT73E1 polypeptide, and/or a UGT76E12 polypeptide; the polypeptide capable of beta-1,2-glycosylation of the C2′ and/or beta-1,3-glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (that is, examples of glycosyl-position glycosylation) activity on a steviol glycoside comprises a UGT73C6 polypeptide, a CaUGT3 polypeptide, a UN32491 polypeptide, and/or a UN1671 polypeptide; and/or the polypeptide is capable of glycosylating a steviol precursor at its C-19 carboxyl or C-19 hydroxyl position comprises a UGT73C1 polypeptide, a UGT73C3 polypeptide, a UGT73C5 polypeptide, a UGT73C6 polypeptide, a UGT73E1 polypeptide, a UGT75B1 polypeptide, a UGT75L6 polypeptide, a UGT76E12 polypeptide, a Olel polypeptide, a UGT5 polypeptide, a SA Gtase, a UDPG1 polypeptide, a UGT74F1 polypeptide, a UGT75D1 polypeptide, a UGT84B2 polypeptide, and/or a UGT74F2-like UGT polypeptide.
In some embodiments of the method for producing one or more steviol glycosides and/or glycosylated steviol precursors, or a composition thereof as disclosed herein by whole cell bioconversion of a plant-derived or synthetic steviol glycoside precursor or a plant-derived or synthetic steviol precursor in a cell culture medium of a recombinant host cell described herein, the UGT73C1 polypeptide comprises a polypeptide having at least 60% identity to an amino acid sequence set forth in SEQ ID NO:127, the UGT73C3 polypeptide comprises a polypeptide having at least 60% identity to an amino acid sequence set forth in SEQ ID NO:133, the UGT73C5 polypeptide comprises a polypeptide having at least 60% identity to an amino acid sequence set forth in SEQ ID NO:135, the UGT73C6 polypeptide comprises a polypeptide having at least 60% identity to an amino acid sequence set forth in SEQ ID NO:137, the UGT73E1 polypeptide comprises a polypeptide having at least 50% identity to an amino acid sequence set forth in SEQ ID NO:141, the UGT75B1 polypeptide comprises a polypeptide having at least 50% sequence identity to an amino acid sequence set forth in SEQ ID NO:145, the UGT75L6 polypeptide comprises a polypeptide having at least 60% sequence identity to an amino acid sequence set forth in SEQ ID NO:147, the UGT76E12 polypeptide comprises a polypeptide having at least 60% sequence identity to an amino acid sequence set forth in SEQ ID NO:153, the Olel polypeptide comprises a polypeptide having at least 55% identity to an amino acid sequence set forth in SEQ ID NO:177, the UGT5 polypeptide comprises a polypeptide having at least 65% identity to an amino acid sequence set forth in SEQ ID NO:181, the SA Gtase polypeptide comprises a polypeptide having at least 55% identity to an amino acid sequence set forth in SEQ ID NO:183, the UDPG1 polypeptide comprises a polypeptide having at least 50% sequence identity to an amino acid sequence set forth in SEQ ID NO:185, the UN1671 polypeptide comprises a polypeptide having at least 45% identity to an amino acid sequence set forth in SEQ ID NO:201, the UGT74F1 polypeptide comprises a polypeptide having at least 50% sequence identity to an amino acid sequence set forth in SEQ ID NO:203, the UGT75D1 polypeptide comprises a polypeptide having at least 50% sequence identity to an amino acid sequence set forth in SEQ ID NO:205, the UGT84B2 polypeptide comprises a polypeptide having at least 40% sequence identity to an amino acid sequence set forth in SEQ ID NO:207, the UGT74F2-like UGT polypeptide comprises a polypeptide having at least 55% identity to an amino acid sequence set forth in SEQ ID NO:211, the UGT73C7 polypeptide comprises a polypeptide having at least 60% identity to an amino acid sequence set forth in SEQ ID NO:139, the CaUGT3 polypeptide comprises a polypeptide having at least 50% identity to an amino acid sequence set forth in SEQ ID NO:169, or the UN32491 polypeptide comprises a polypeptide having at least 50% identity to an amino acid sequence set forth in SEQ ID NO:199.
In some embodiments, a polypeptide, e.g., a UGT polypeptide, can be displayed on the surface of the recombinant host cells disclosed herein by fusing it with anchoring motifs.
In some embodiments, the cell is permeabilized to take up a substrate to be modified or to excrete a modified product. In some embodiments, a permeabilizing agent can be added to aid the feedstock entering into the host and product getting out. In some embodiments, the cells are permeabilized with a solvent such as toluene, or with a detergent such as Triton-X or Tween. In some embodiments, the cells are permeabilized with a surfactant, for example a cationic surfactant such as cetyltrimethylammonium bromide (CTAB). In some embodiments, the cells are permeabilized with periodic mechanical shock such as electroporation or a slight osmotic shock. For example, a crude lysate of the cultured microorganism can be centrifuged to obtain a supernatant. The resulting supernatant can then be applied to a chromatography column, e.g., a C18 column, and washed with water to remove hydrophilic compounds, followed by elution of the compound(s) of interest with a solvent such as methanol. The compound(s) can then be further purified by preparative HPLC. See also, WO 2009/140394.
In some embodiments, steviol, one or more steviol glycoside precursors, and/or one or more steviol glycosides are produced by co-culturing of two or more hosts. In some embodiments, one or more hosts, each expressing one or more enzymes involved in the steviol glycoside pathway, produce steviol, one or more steviol glycoside precursors, and/or one or more steviol glycosides. For example, a host expressing a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP; a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP; a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from ent-kaurene; a gene encoding a polypeptide capable of reducing cytochrome P450 complex; a gene encoding a polypeptide capable of synthesizing steviol from ent-kaurenoic acid; and/or a gene encoding a bifunctional polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP and synthesizing ent-kaurene from ent-copalyl diphosphate and a host expressing a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl position; a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl position; and/or a gene encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, produce one or more steviol glycosides.
In some embodiments, a recombinant host comprising a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl position, e.g., a UGT73C1 polypeptide, a UGT73C3 polypeptide, a UGT73C5 polypeptide, a UGT73C6 polypeptide, a UGT73E1 polypeptide, a UGT75B1 polypeptide, a UGT75L6 polypeptide, a Olel polypeptide, a UGT5 polypeptide, a SA Gtase polypeptide, a UDPG1 polypeptide, a UN1671 polypeptide, a UGT74F1 polypeptide, a UGT84B2 polypeptide, and/or a UGT74F2-like UGT polypeptide further comprises a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its 0-13 hydroxyl position (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:7); a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:9); a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl position (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:4); and/or a gene encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:16). In certain such embodiments, the recombinant host cell further comprises a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:20); a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:40); a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl diphosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:52); a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from ent-kaurene (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:60 or SEQ ID NO:117); a gene encoding a polypeptide capable of reducing cytochrome P450 complex (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:78, SEQ ID NO:86, or SEQ ID NO:92); and/or a gene encoding a polypeptide capable of synthesizing steviol from ent-kaurenoic acid (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:94).
In some embodiments, a recombinant host comprising a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl position, e.g., a UGT73C1 polypeptide, a UGT73C3 polypeptide, a UGT73C5 polypeptide, a UGT73C6 polypeptide, a UGT73C7 polypeptide, a UGT73E1 polypeptide, and/or a UGT76E12 polypeptide further comprises a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl position (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:7); a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:9); a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl position (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:4); and/or a gene encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:16). In certain such embodiments, the recombinant host cell further comprises a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:20); a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:40); a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl diphosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:52); a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from ent-kaurene (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:60 or SEQ ID NO:117); a gene encoding a polypeptide capable of reducing cytochrome P450 complex (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:78, SEQ ID NO:86, or SEQ ID NO:92); and/or a gene encoding a polypeptide capable of synthesizing steviol from ent-kaurenoic acid (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:94).
In some embodiments, a recombinant host comprising a gene encoding a polypeptide capable of beta-1,2-glycosylation of the C2′ and/or beta-1,3-glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (that is, examples of glycosyl-position glycosylation), e.g., a UGT73C6 polypeptide, a CaUGT3 polypeptide, a UN32491 polypeptide, and/or a UN1671 polypeptide further comprises a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl position (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:7); a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:9); a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl position (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:4); and/or a gene encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:16). In certain such embodiments, the recombinant host cell further comprises a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:20); a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:40); a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl diphosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:52); a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from ent-kaurene (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:60 or SEQ ID NO:117); a gene encoding a polypeptide capable of reducing cytochrome P450 complex (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:78, SEQ ID NO:86, or SEQ ID NO:92); and/or a gene encoding a polypeptide capable of synthesizing steviol from ent-kaurenoic acid (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:94).
In some embodiments, a recombinant host comprising a gene encoding a polypeptide capable of glycosylating a steviol precursor at its C-19 carboxyl or C-19 hydroxyl position, e.g., a UGT73C1 polypeptide, a UGT73C3 polypeptide, a UGT73C5 polypeptide, a UGT73C6 polypeptide, a UGT73E1 polypeptide, a UGT75B1 polypeptide, a UGT75L6 polypeptide, a UGT76E12 polypeptide, a Olel polypeptide, a UGT5 polypeptide, a SA Gtase, a UDPG1 polypeptide, a UGT74F1 polypeptide, a UGT75D1 polypeptide, a UGT84B2 polypeptide, and/or a UGT74F2-like UGT polypeptide further comprises a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl position (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:7); a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:9); a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl position (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:4); and/or a gene encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:16). In certain such embodiments, the recombinant host cell further comprises a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:20); a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:40); a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl diphosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:52); a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenol from ent-kaurene (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:60 or SEQ ID NO:117); a gene encoding a polypeptide capable of reducing cytochrome P450 complex (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:78, SEQ ID NO:86, or SEQ ID NO:92); and/or a gene encoding a polypeptide capable of synthesizing steviol from ent-kaurenoic acid (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:94).
In some embodiments, a recombinant host comprising a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl position, e.g., a SA Gtase (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:183) further comprises a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl position (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:7); a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:9); a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl position (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:4); and/or a gene encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:16). In certain such embodiments, the recombinant host cell further comprises a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:20); a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:40); a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl diphosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:52); a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from ent-kaurene (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:60 or SEQ ID NO:117); a gene encoding a polypeptide capable of reducing cytochrome P450 complex (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:78, SEQ ID NO:86, or SEQ ID NO:92); and/or a gene encoding a polypeptide capable of synthesizing steviol from ent-kaurenoic acid (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:94).
In some aspects, expression of SA Gtase (SEQ ID NO:182, SEQ ID NO:183) in S. cerevisiae comprising one or more copies of a recombinant gene encoding a GGPPS polypeptide (e.g., SEQ ID NO:19, SEQ ID NO:20), a recombinant gene encoding a truncated CDPS polypeptide (e.g., SEQ ID NO:39, SEQ ID NO:40), a recombinant gene encoding a KS polypeptide (e.g., SEQ ID NO:51, SEQ ID NO:52), a recombinant gene encoding a KO polypeptide (e.g., SEQ ID NO:59, SEQ ID NO:60), a recombinant gene encoding an ATR2 polypeptide (e.g., SEQ ID NO:91, SEQ ID NO:92), a recombinant gene encoding an EUGT11 polypeptide (e.g., SEQ ID NO:14/SEQ ID NO:15, SEQ ID NO:16), a recombinant gene encoding a KAH polypeptide (e.g., SEQ ID NO:93, SEQ ID NO:94), a recombinant gene encoding a CPR8 polypeptide (e.g., SEQ ID NO:85, SEQ ID NO:86), a recombinant gene encoding a UGT85C2 polypeptide (e.g., SEQ ID NO:5/SEQ ID NO:6/SEQ ID NO:149, SEQ ID NO:7) or a UGT85C2 variant (or functional homolog) of SEQ ID NO:7, a recombinant gene encoding a UGT74G1 polypeptide (e.g., SEQ ID NO:3, SEQ ID NO:4) of a UGT74G1 variant (or functional homolog) of SEQ ID NO:4, a recombinant gene encoding a UGT76G1 polypeptide (e.g., SEQ ID NO:8, SEQ ID NO:9) or a UGT76G1 variant (or functional homolog) of SEQ ID NO:9, and a recombinant gene encoding a UGT91D2e polypeptide (e.g., SEQ ID NO:10, SEQ ID NO:11) and/or a UGT91D2e variant (or functional homolog) of SEQ ID NO:11 such as a UGT91D2e-b (SEQ ID NO:12, SEQ ID NO:13) polypeptide results in increased ent-kaurenoic acid+2Glc (#7), ent-kaurenoic acid+3Glc (isomer 1), ent-kaurenoic acid+3Glc (isomer 2), 13-SMG, RebA, RebB, Steviol+4Glc (#36), Steviol+6Glc (isomer 1), Steviol+7Glc (isomer 2), and/or ent-Kaurenol+3Glc (isomer 1 and/or isomer 2). See, Example 4.
In some embodiments, a steviol glycoside and/or glycoside of a steviol precursor, or a composition thereof produced in vivo, in vitro, or by whole cell bioconversion comprises fewer contaminants or less of any particular contaminant than a stevia extract from, inter alia, a stevia plant. Contaminants can include plant-derived compounds that contribute to off-flavors. Potential contaminants include pigments, lipids, proteins, phenolics, saccharides, spathulenol and other sesquiterpenes, labdane diterpenes, monoterpenes, decanoic acid, 8,11,14-eicosatrienoic acid, 2-methyloctadecane, pentacosane, octacosane, tetracosane, octadecanol, stigmasterol, β-sitosterol, α-amyrin, β-amyrin, lupeol, β-amryin acetate, pentacyclic triterpenes, centauredin, quercitin, epi-alpha-cadinol, carophyllenes and derivatives, beta-pinene, beta-sitosterol, and gibberellin.
As used herein, the terms “detectable amount,” “detectable concentration,” “measurable amount,” and “measurable concentration” refer to a level of steviol glycosides measured in AUC, μM/OD600, mg/L, μM, or mM. Steviol glycoside production (i.e., total, supernatant, and/or intracellular steviol glycoside levels) can be detected and/or analyzed by techniques generally available to one skilled in the art, for example, but not limited to, liquid chromatography-mass spectrometry (LC-MS), thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), ultraviolet-visible spectroscopy/spectrophotometry (UV-Vis), mass spectrometry (MS), and NMR.
As used herein, the term “undetectable concentration” refers to a level of a compound that is too low to be measured and/or analyzed by techniques such as TLC, HPLC, UV-Vis, MS, or NMR. In some embodiments, a compound of an “undetectable concentration” is not present in a steviol glycoside or steviol glycoside precursor composition.
As used herein, the terms “or” and “and/or” is utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and/or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” In some embodiments, “and/or” is used to refer to the exogenous nucleic acids that a recombinant cell comprises, wherein a recombinant cell comprises one or more exogenous nucleic acids selected from a group. In some embodiments, “and/or” is used to refer to production of steviol glycosides and/or steviol glycoside precursors. In some embodiments, “and/or” is used to refer to production of steviol glycosides, wherein one or more steviol glycosides are produced. In some embodiments, “and/or” is used to refer to production of steviol glycosides, wherein one or more steviol glycosides are produced through one or more of the following steps: culturing a recombinant microorganism, synthesizing one or more steviol glycosides in a recombinant microorganism, and/or isolating one or more steviol glycosides.
Functional homologs of the polypeptides described above are also suitable for use in producing steviol glycosides in a recombinant host. A functional homolog is a polypeptide that has sequence similarity to a reference polypeptide, and that carries out one or more of the biochemical or physiological function(s) of the reference polypeptide. A functional homolog and the reference polypeptide can be a natural occurring polypeptide, and the sequence similarity can be due to convergent or divergent evolutionary events. As such, functional homologs are sometimes designated in the literature as homologs, or orthologs, or paralogs. Variants of a naturally occurring functional homolog, such as polypeptides encoded by mutants of a wild type coding sequence, can themselves be functional homologs. Functional homologs can also be created via site-directed mutagenesis of the coding sequence for a polypeptide, or by combining domains from the coding sequences for different naturally-occurring polypeptides (“domain swapping”). Techniques for modifying genes encoding functional polypeptides described herein are known and include, inter alia, directed evolution techniques, site-directed mutagenesis techniques and random mutagenesis techniques, and can be useful to increase specific activity of a polypeptide, alter substrate specificity, alter expression levels, alter subcellular location, or modify polypeptide-polypeptide interactions in a desired manner. Such modified polypeptides are considered functional homologs. The term “functional homolog” is sometimes applied to the nucleic acid that encodes a functionally homologous polypeptide.
Functional homologs can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologs of steviol glycoside biosynthesis polypeptides. Sequence analysis can involve BLAST, Reciprocal BLAST, or PSI-BLAST analysis of non-redundant databases using a UGT amino acid sequence as the reference sequence. Amino acid sequence is, in some instances, deduced from the nucleotide sequence. Those polypeptides in the database that have greater than 40% sequence identity are candidates for further evaluation for suitability as a steviol glycoside biosynthesis polypeptide. Amino acid sequence similarity allows for conservative amino acid substitutions, such as substitution of one hydrophobic residue for another or substitution of one polar residue for another. If desired, manual inspection of such candidates can be carried out in order to narrow the number of candidates to be further evaluated. Manual inspection can be performed by selecting those candidates that appear to have domains present in steviol glycoside biosynthesis polypeptides, e.g., conserved functional domains. In some embodiments, nucleic acids and polypeptides are identified from transcriptome data based on expression levels rather than by using BLAST analysis.
Conserved regions can be identified by locating a region within the primary amino acid sequence of a steviol glycoside biosynthesis polypeptide that is a repeated sequence, forms some secondary structure (e.g., helices and beta sheets), establishes positively or negatively charged domains, or represents a protein motif or domain. See, e.g., the Pfam web site describing consensus sequences for a variety of protein motifs and domains on the World Wide Web at sanger.ac.uk/Software/Pfam/and pfam.janelia.org/. The information included at the Pfam database is described in Sonnhammer et al., Nucl. Acids Res., 26:320-322 (1998); Sonnhammer et al., Proteins, 28:405-420 (1997); and Bateman et al., Nucl. Acids Res., 27:260-262 (1999). Conserved regions also can be determined by aligning sequences of the same or related polypeptides from closely related species. Closely related species preferably are from the same family. In some embodiments, alignment of sequences from two different species is adequate to identify such homologs.
Typically, polypeptides that exhibit at least about 40% amino acid sequence identity are useful to identify conserved regions. Conserved regions of related polypeptides exhibit at least 45% amino acid sequence identity (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% amino acid sequence identity). In some embodiments, a conserved region exhibits at least 92%, 94%, 96%, 98%, or 99% amino acid sequence identity.
For example, polypeptides suitable for producing steviol in a recombinant host include functional homologs of UGTs.
Methods to modify the substrate specificity of, for example, a UGT, are known to those skilled in the art, and include without limitation site-directed/rational mutagenesis approaches, random directed evolution approaches and combinations in which random mutagenesis/saturation techniques are performed near the active site of the enzyme. For example see Osmani et al., 2009, Phytochemistry 70: 325-347.
A candidate sequence typically has a length that is from 80% to 200% of the length of the reference sequence, e.g., 82, 85, 87, 89, 90, 93, 95, 97, 99, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, or 200% of the length of the reference sequence. A functional homolog polypeptide typically has a length that is from 95% to 105% of the length of the reference sequence, e.g., 90, 93, 95, 97, 99, 100, 105, 110, 115, or 120% of the length of the reference sequence, or any range between. A % identity for any candidate nucleic acid or polypeptide relative to a reference nucleic acid or polypeptide can be determined as follows. A reference sequence (e.g., a nucleic acid sequence or an amino acid sequence described herein) is aligned to one or more candidate sequences using the computer program Clustal Omega (version 1.2.1, default parameters), which allows alignments of nucleic acid or polypeptide sequences to be carried out across their entire length (global alignment). Chenna et al., 2003, Nucleic Acids Res. 31(13):3497-500.
ClustalW calculates the best match between a reference and one or more candidate sequences, and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a reference sequence, a candidate sequence, or both, to maximize sequence alignments. For fast pairwise alignment of nucleic acid sequences, the following default parameters are used: word size: 2; window size: 4; scoring method: % age; number of top diagonals: 4; and gap penalty: 5. For multiple alignment of nucleic acid sequences, the following parameters are used: gap opening penalty: 10.0; gap extension penalty: 5.0; and weight transitions: yes. For fast pairwise alignment of protein sequences, the following parameters are used: word size: 1; window size: 5; scoring method:% age; number of top diagonals: 5; gap penalty: 3. For multiple alignment of protein sequences, the following parameters are used: weight matrix: blosum; gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps: on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, and Lys; residue-specific gap penalties: on. The ClustalW output is a sequence alignment that reflects the relationship between sequences. ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher site on the World Wide Web (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at the European Bioinformatics Institute site on the World Wide Web (ebi.ac.uk/clustalw).
To determine a % identity of a candidate nucleic acid or amino acid sequence to a reference sequence, the sequences are aligned using Clustal Omega, the number of identical matches in the alignment is divided by the length of the reference sequence, and the result is multiplied by 100. It is noted that the % identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.
It will be appreciated that functional UGT proteins can include additional amino acids that are not involved in the enzymatic activities carried out by the enzymes. In some embodiments, UGT proteins are fusion proteins. The terms “chimera,” “fusion polypeptide,” “fusion protein,” “fusion enzyme,” “fusion construct,” “chimeric protein,” “chimeric polypeptide,” “chimeric construct,” and “chimeric enzyme” can be used interchangeably herein to refer to proteins engineered through the joining of two or more genes that code for different proteins. In some embodiments, a nucleic acid sequence encoding a UGT polypeptide can include a tag sequence that encodes a “tag” designed to facilitate subsequent manipulation (e.g., to facilitate purification or detection), secretion, or localization of the encoded polypeptide. Tag sequences can be inserted in the nucleic acid sequence encoding the polypeptide such that the encoded tag is located at either the carboxyl or amino terminus of the polypeptide. Non-limiting examples of encoded tags include green fluorescent protein (GFP), human influenza hemagglutinin (HA), glutathione S transferase (GST), polyhistidine-tag (HIS tag), and Flag™ tag (Kodak, New Haven, Conn.). Other examples of tags include a chloroplast transit peptide, a mitochondrial transit peptide, an amyloplast peptide, signal peptide, or a secretion tag.
In some embodiments, a fusion protein is a protein altered by domain swapping. As used herein, the term “domain swapping” is used to describe the process of replacing a domain of a first protein with a domain of a second protein. In some embodiments, the domain of the first protein and the domain of the second protein are functionally identical or functionally similar. In some embodiments, the structure and/or sequence of the domain of the second protein differs from the structure and/or sequence of the domain of the first protein. In some embodiments, a UGT polypeptide is altered by domain swapping.
In some embodiments, a fusion protein is a protein altered by circular permutation, which consists in the covalent attachment of the ends of a protein that would be opened elsewhere afterwards. Thus, the order of the sequence is altered without causing changes in the amino acids of the protein. In some embodiments, a targeted circular permutation can be produced, for example but not limited to, by designing a spacer to join the ends of the original protein. Once the spacer has been defined, there are several possibilities to generate permutations through generally accepted molecular biology techniques, for example but not limited to, by producing concatemers by means of PCR and subsequent amplification of specific permutations inside the concatemer or by amplifying discrete fragments of the protein to exchange to join them in a different order. The step of generating permutations can be followed by creating a circular gene by binding the fragment ends and cutting back at random, thus forming collections of permutations from a unique construct.
A recombinant gene encoding a polypeptide described herein comprises the coding sequence for that polypeptide, operably linked in sense orientation to one or more regulatory regions suitable for expressing the polypeptide. Because many microorganisms are capable of expressing multiple gene products from a polycistronic mRNA, multiple polypeptides can be expressed under the control of a single regulatory region for those microorganisms, if desired. A coding sequence and a regulatory region are considered to be operably linked when the regulatory region and coding sequence are positioned so that the regulatory region is effective for regulating transcription or translation of the sequence. Typically, the translation initiation site of the translational reading frame of the coding sequence is positioned between one and about fifty nucleotides downstream of the regulatory region for a monocistronic gene.
In many cases, the coding sequence for a polypeptide described herein is identified in a species other than the recombinant host, i.e., is a heterologous nucleic acid. Thus, if the recombinant host is a microorganism, the coding sequence can be from other prokaryotic or eukaryotic microorganisms, from plants or from animals. In some case, however, the coding sequence is a sequence that is native to the host and is being reintroduced into that organism.
A native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct. In addition, stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found. “Regulatory region” refers to a nucleic acid having nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of a transcription or translation product. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5′ and 3′ untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, introns, and combinations thereof. A regulatory region typically comprises at least a core (basal) promoter. A regulatory region also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR). A regulatory region is operably linked to a coding sequence by positioning the regulatory region and the coding sequence so that the regulatory region is effective for regulating transcription or translation of the sequence. For example, to operably link a coding sequence and a promoter sequence, the translation initiation site of the translational reading frame of the coding sequence is typically positioned between one and about fifty nucleotides downstream of the promoter. A regulatory region can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the transcription start site.
The choice of regulatory regions to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and preferential expression during certain culture stages. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning regulatory regions relative to the coding sequence. It will be understood that more than one regulatory region may be present, e.g., introns, enhancers, upstream activation regions, transcription terminators, and inducible elements.
One or more genes can be combined in a recombinant nucleic acid construct in “modules” useful for a discrete aspect of steviol and/or steviol glycoside production. Combining a plurality of genes in a module, particularly a polycistronic module, facilitates the use of the module in a variety of species. For example, a steviol biosynthesis gene cluster, or a UGT gene cluster, can be combined in a polycistronic module such that, after insertion of a suitable regulatory region, the module can be introduced into a wide variety of species. As another example, a UGT gene cluster can be combined such that each UGT coding sequence is operably linked to a separate regulatory region, to form a UGT module. Such a module can be used in those species for which monocistronic expression is necessary or desirable. In addition to genes useful for steviol or steviol glycoside production, a recombinant construct typically also contains an origin of replication, and one or more selectable markers for maintenance of the construct in appropriate species.
It will be appreciated that because of the degeneracy of the genetic code, a number of nucleic acids can encode a particular polypeptide; i.e., for many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid. Thus, codons in the coding sequence for a given polypeptide can be modified such that optimal expression in a particular host is obtained, using appropriate codon bias tables for that host (e.g., microorganism). As isolated nucleic acids, these modified sequences can exist as purified molecules and can be incorporated into a vector or a virus for use in constructing modules for recombinant nucleic acid constructs.
In some cases, it is desirable to inhibit one or more functions of an endogenous polypeptide in order to divert metabolic intermediates towards steviol or steviol glycoside biosynthesis. For example, it may be desirable to downregulate synthesis of sterols in a yeast strain in order to further increase steviol or steviol glycoside production, e.g., by downregulating squalene epoxidase. As another example, it may be desirable to inhibit degradative functions of certain endogenous gene products, e.g., glycohydrolases that remove glucose moieties from secondary metabolites or phosphatases as discussed herein. In such cases, a nucleic acid that overexpresses the polypeptide or gene product may be included in a recombinant construct that is transformed into the strain. Alternatively, mutagenesis can be used to generate mutants in genes for which it is desired to increase or enhance function.
One aspect of the disclosure is an isolated nucleic acid molecule encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl position or a catalytically active portion thereof. The nucleic acid is cDNA. In some embodiments, the encoded polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl position or the catalytically active portion thereof comprises a a UGT73C1 polypeptide, a UGT73C3 polypeptide, a UGT73C5 polypeptide, a UGT73C6 polypeptide, a UGT73E1 polypeptide, a UGT75B1 polypeptide, a UGT75L6 polypeptide, a Olel polypeptide, a UGT5 polypeptide, a SA Gtase polypeptide, a UDPG1 polypeptide, a UN1671 polypeptide, a UGT74F1 polypeptide, a UGT84B2 polypeptide, or a UGT74F2-like UGT polypeptide. In some embodiments, the encoded polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl position or the catalytically active portion thereof comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:127, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:141, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:177, SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:207, or SEQ ID NO:211.
Another aspect of the disclosure is an isolated nucleic acid molecule encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl position or a catalytically active portion thereof. In some embodiments, the encoded polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl position or the catalytically active portion thereof comprises a UGT73C1 polypeptide, a UGT73C3 polypeptide, a UGT73C5 polypeptide, a UGT73C6 polypeptide, a UGT73C7 polypeptide, a UGT73E1 polypeptide, or a UGT76E12 polypeptide. In some embodiments, the encoded polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl position or the catalytically active portion thereof comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:127, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, or SEQ ID NO:153.
Another aspect of the disclosure is an isolated nucleic acid molecule encoding a polypeptide capable of beta-1,2-glycosylation of the C2′ and/or beta-1,3-glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside or a catalytically active portion thereof. The nucleic acid is cDNA. In some embodiments, the encoded polypeptide capable of beta-1,2-glycosylation of the C2′ and/or beta-1,3-glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside or the catalytically active portion thereof comprises a UGT73C6 polypeptide, a CaUGT3 polypeptide, a UN32491 polypeptide, or a UN1671 polypeptide. In some embodiments, the encoded polypeptide capable of beta-1,2-glycosylation of the C2′ and/or beta-1,3-glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside or the catalytically active portion thereof comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 137, SEQ ID NO:169, SEQ ID NO:199, or SEQ ID NO:201.
Another aspect of the disclosure is an isolated nucleic acid molecule encoding a polypeptide capable of glycosylating a steviol precursor at its C-19 carboxyl or C-19 hydroxyl position or a catalytically active portion thereof. The nucleic acid is cDNA. In some embodiments, the encoded polypeptide capable of glycosylating a steviol precursor at its C-19 carboxyl or C-19 hydroxyl position or the catalytically active portion thereof comprises a UGT73C1 polypeptide, a UGT73C3 polypeptide, a UGT73C5 polypeptide, a UGT73C6 polypeptide, a UGT73E1 polypeptide, a UGT75B1 polypeptide, a UGT75L6 polypeptide, a UGT76E12 polypeptide, a Olel polypeptide, a UGT5 polypeptide, a SA Gtase, a UDPG1 polypeptide, a UGT74F1 polypeptide, a UGT75D1 polypeptide, a UGT84B2 polypeptide, or a UGT74F2-like UGT polypeptide. In some embodiments, the encoded polypeptide capable of glycosylating a steviol precursor at its C-19 carboxyl or C-19 hydroxyl position or the catalytically active portion thereof comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 127, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:141, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:153, SEQ ID NO:177, SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, or SEQ ID NO:211.
Recombinant hosts can be used to express polypeptides for the producing steviol glycosides, including mammalian, insect, plant, and algal cells. A number of prokaryotes and eukaryotes are also suitable for use in constructing the recombinant microorganisms described herein, e.g., gram-negative bacteria, yeast, and fungi. A species and strain selected for use as a steviol glycoside production strain is first analyzed to determine which production genes are endogenous to the strain and which genes are not present. Genes for which an endogenous counterpart is not present in the strain are advantageously assembled in one or more recombinant constructs, which are then transformed into the strain in order to supply the missing function(s).
Typically, the recombinant microorganism is grown in a fermenter at a temperature(s) for a period of time, wherein the temperature and period of time facilitate the production of a steviol glycoside. The constructed and genetically engineered microorganisms provided by the invention can be cultivated using conventional fermentation processes, including, inter alia, chemostat, batch, fed-batch cultivations, semi-continuous fermentations such as draw and fill, continuous perfusion fermentation, and continuous perfusion cell culture. Depending on the particular microorganism used in the method, other recombinant genes such as isopentenyl biosynthesis genes and terpene synthase and cyclase genes may also be present and expressed. Levels of substrates and intermediates, e.g., isopentenyl diphosphate, dimethylallyl diphosphate, GGPP, ent-kaurene and ent-kaurenoic acid, can be determined by extracting samples from culture media for analysis according to published methods.
Carbon sources of use in the instant method include any molecule that can be metabolized by the recombinant host cell to facilitate growth and/or production of the steviol glycosides. Examples of suitable carbon sources include, but are not limited to, sucrose (e.g., as found in molasses), fructose, xylose, ethanol, glycerol, glucose, cellulose, starch, cellobiose or other glucose-comprising polymer. In embodiments employing yeast as a host, for example, carbons sources such as sucrose, fructose, xylose, ethanol, glycerol, and glucose are suitable. The carbon source can be provided to the host organism throughout the cultivation period or alternatively, the organism can be grown for a period of time in the presence of another energy source, e.g., protein, and then provided with a source of carbon only during the fed-batch phase.
After the recombinant microorganism has been grown in culture for the period of time, wherein the temperature and period of time facilitate the production of a steviol glycoside, steviol and/or one or more steviol glycosides can then be recovered from the culture using various techniques known in the art. In some embodiments, a permeabilizing agent can be added to aid the feedstock entering into the host and product getting out. For example, a crude lysate of the cultured microorganism can be centrifuged to obtain a supernatant. The resulting supernatant can then be applied to a chromatography column, e.g., a C-18 column, and washed with water to remove hydrophilic compounds, followed by elution of the compound(s) of interest with a solvent such as methanol. The compound(s) can then be further purified by preparative HPLC. See also, WO 2009/140394.
It will be appreciated that the various genes and modules discussed herein can be present in two or more recombinant hosts rather than a single host. When a plurality of recombinant hosts is used, they can be grown in a mixed culture to accumulate steviol and/or steviol glycosides.
Alternatively, the two or more hosts each can be grown in a separate culture medium and the product of the first culture medium, e.g., steviol, can be introduced into second culture medium to be converted into a subsequent intermediate, or into an end product such as, for example, RebA. The product produced by the second, or final host is then recovered. It will also be appreciated that in some embodiments, a recombinant host is grown using nutrient sources other than a culture medium and utilizing a system other than a fermenter.
Exemplary prokaryotic and eukaryotic species are described in more detail below. However, it will be appreciated that other species can be suitable. For example, suitable species can be in a genus such as Agaricus, Aspergillus, Bacillus, Candida, Corynebacterium, Eremothecium, Escherichia, Fusarium/Gibberella, Kluyveromyces, Laetiporus, Lentinus, Phaffia, Phanerochaete, Pichia, Physcomitrella, Rhodoturula, Saccharomyces, Schizosaccharomyces, Sphaceloma, Xanthophyllomyces or Yarrowia. Exemplary species from such genera include Lentinus tigrinus, Laetiporus sulphureus, Phanerochaete chrysosporium, Pichia pastoris, Cyberlindnera jadinii, Physcomitrella patens, Rhodoturula glutinis, Rhodoturula mucilaginosa, Phaffia rhodozyma, Xanthophyllomyces dendrorhous, Fusarium fujikuroi/Gibberella fujikuroi, Candida utilis, Candida glabrata, Candida albicans, and Yarrowia lipolytica.
In some embodiments, a microorganism can be a prokaryote such as Escherichia bacteria cells, for example, Escherichia coli cells; Lactobacillus bacteria cells; Lactococcus bacteria cells; Comebacterium bacteria cells; Acetobacter bacteria cells; Acinetobacter bacteria cells; or Pseudomonas bacterial cells.
In some embodiments, a microorganism can be an Ascomycete such as Gibberella fujikuroi, Kluyveromyces lactis, Schizosaccharomyces pombe, Aspergillus niger, Yarrowia lipolytica, Ashbya gossypii, or S. cerevisiae.
In some embodiments, a microorganism can be an algal cell such as Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp., Undaria pinnatifida, Sargassum, Laminaria japonica, Scenedesmus almeriensis species.
In some embodiments, a microorganism can be a cyanobacterial cell such as Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp., Undaria pinnatifida, Sargassum, Laminaria japonica, Scenedesmus almeriensis.
Saccharomyces is a widely used chassis organism in synthetic biology, and can be used as the recombinant microorganism platform. For example, there are libraries of mutants, plasmids, detailed computer models of metabolism and other information available for S. cerevisiae, allowing for rational design of various modules to enhance product yield. Methods are known for making recombinant microorganisms.
Aspergillus species such as A. oryzae, A. niger and A. sojae are widely used microorganisms in food production and can also be used as the recombinant microorganism platform. Nucleotide sequences are available for genomes of A. nidulans, A. fumigatus, A. oryzae, A. clavatus, A. flavus, A. niger, and A. terreus, allowing rational design and modification of endogenous pathways to enhance flux and increase product yield. Metabolic models have been developed for Aspergillus, as well as transcriptomic studies and proteomics studies. A. niger is cultured for the industrial production of a number of food ingredients such as citric acid and gluconic acid, and thus species such as A. niger are generally suitable for producing steviol glycosides.
E. coli
E. coli, another widely used platform organism in synthetic biology, can also be used as the recombinant microorganism platform. Similar to Saccharomyces, there are libraries of mutants, plasmids, detailed computer models of metabolism and other information available for E. coli, allowing for rational design of various modules to enhance product yield. Methods similar to those described above for Saccharomyces can be used to make recombinant E. coli microorganisms.
Agaricus, Gibberella, and Phanerochaete spp. can be useful because they are known to produce large amounts of isoprenoids in culture. Thus, the terpene precursors for producing large amounts of steviol glycosides are already produced by endogenous genes. Thus, modules comprising recombinant genes for steviol glycoside biosynthesis polypeptides can be introduced into species from such genera without the necessity of introducing mevalonate or MEP pathway genes.
Arxula adeninivorans (Blastobotrys adeninivorans)
Arxula adeninivorans is dimorphic yeast (it grows as budding yeast like the baker's yeast up to a temperature of 42° C., above this threshold it grows in a filamentous form) with unusual biochemical characteristics. It can grow on a wide range of substrates and can assimilate nitrate. It has successfully been applied to the generation of strains that can produce natural plastics or the development of a biosensor for estrogens in environmental samples.
Yarrowia lipolytica
Yarrowia lipolytica is dimorphic yeast (see Arxula adeninivorans) and belongs to the family Hemiascomycetes. The entire genome of Yarrowia lipolytica is known. Yarrowia species is aerobic and considered to be non-pathogenic. Yarrowia is efficient in using hydrophobic substrates (e.g. alkanes, fatty acids, oils) and can grow on sugars. It has a high potential for industrial applications and is an oleaginous microorgamism. Yarrowia lipolyptica can accumulate lipid content to approximately 40% of its dry cell weight and is a model organism for lipid accumulation and remobilization. See e.g., Nicaud, 2012, Yeast 29(10):409-18; Beopoulos et al., 2009, Biochimie 91(6):692-6; Bankar et al., 2009, Appl Microbiol Biotechnol. 84(5):847-65.
Rhodotorula is unicellular, pigmented yeast. The oleaginous red yeast, Rhodotorula glutinis, has been shown to produce lipids and carotenoids from crude glycerol (Saenge et al., 2011, Process Biochemistry 46(1):210-8). Rhodotorula toruloides strains have been shown to be an efficient fed-batch fermentation system for improved biomass and lipid productivity (Li et al., 2007, Enzyme and Microbial Technology 41:312-7).
Rhodosporidium toruloides
Rhodosporidium toruloides is oleaginous yeast and useful for engineering lipid-production pathways (See e.g. Zhu et al., 2013, Nature Commun. 3:1112; Ageitos et al., 2011, Applied Microbiology and Biotechnology 90(4):1219-27).
Candida boidinii
Candida boidinii is methylotrophic yeast (it can grow on methanol). Like other methylotrophic species such as Hansenula polymorpha and Pichia pastoris, it provides an excellent platform for producing heterologous proteins. Yields in a multigram range of a secreted foreign protein have been reported. A computational method, IPRO, recently predicted mutations that experimentally switched the cofactor specificity of Candida boidinii xylose reductase from NADPH to NADH. See, e.g., Mattanovich et al., 2012, Methods Mol Biol. 824:329-58; Khoury et al., 2009, Protein Sci. 18(10):2125-38.
Hansenula polymorpha (Pichia angusta)
Hansenula polymorpha is methylotrophic yeast (see Candida boidinii). It can furthermore grow on a wide range of other substrates; it is thermo-tolerant and can assimilate nitrate (see also Kluyveromyces lactis). It has been applied to producing hepatitis B vaccines, insulin and interferon alpha-2a for the treatment of hepatitis C, furthermore to a range of technical enzymes. See, e.g., Xu et al., 2014, Virol Sin. 29(6):403-9.
Kluyveromyces lactis
Kluyveromyces lactis is yeast regularly applied to the production of kefir. It can grow on several sugars, most importantly on lactose which is present in milk and whey. It has successfully been applied among others for producing chymosin (an enzyme that is usually present in the stomach of calves) for producing cheese. Production takes place in fermenters on a 40,000 L scale. See, e.g., van Ooyen et al., 2006, FEMS Yeast Res. 6(3):381-92.
Pichia pastoris
Pichia pastoris is methylotrophic yeast (see Candida boidinii and Hansenula polymorpha). It provides an efficient platform for producing foreign proteins. Platform elements are available as a kit and it is worldwide used in academia for producing proteins. Strains have been engineered that can produce complex human N-glycan (yeast glycans are similar but not identical to those found in humans). See, e.g., Piirainen et al., 2014, N Biotechnol. 31(6):532-7.
Physcomitrella mosses, when grown in suspension culture, have characteristics similar to yeast or other fungal cultures. This genera can be used for producing plant secondary metabolites, which can be difficult to produce in other types of cells.
It will be appreciated that the recombinant host cell disclosed herein can comprise a plant cell, comprising a plant cell that is grown in a plant, a mammalian cell, an insect cell, a fungal cell, comprising a yeast cell, wherein the yeast cell is a cell from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, or Candida albicans species or is a Saccharomycete or is a Saccharomyces cerevisiae cell, an algal cell or a bacterial cell, comprising Escherichia cells, Lactobacillus cells, Lactococcus cells, Cornebacterium cells, Acetobacter cells, Acinetobacter cells, or Pseudomonas cells.
Steviol glycosides do not necessarily have equivalent performance in different food systems. It is therefore desirable to have the ability to direct the synthesis to steviol glycoside compositions of choice. Recombinant hosts described herein can produce compositions that are selectively enriched for specific steviol glycosides (e.g., RebD or RebM) and have a consistent taste profile. As used herein, the term “enriched” is used to describe a steviol glycoside composition with an increased proportion of a particular steviol glycoside, compared to a steviol glycoside composition (extract) from a stevia plant. Thus, the recombinant hosts described herein can facilitate the production of compositions that are tailored to meet the sweetening profile desired for a given food product and that have a proportion of each steviol glycoside that is consistent from batch to batch. In some embodiments, hosts described herein do not produce or produce a reduced amount of undesired plant by-products found in Stevia extracts. Thus, steviol glycoside compositions produced by the recombinant hosts described herein are distinguishable from compositions derived from Stevia plants.
The amount of an individual steviol glycoside (e.g., RebA, RebB, RebD, or RebM) accumulated can be from about 1 to about 7,000 mg/L, e.g., about 1 to about 10 mg/L, about 3 to about 10 mg/L, about 5 to about 20 mg/L, about 10 to about 50 mg/L, about 10 to about 100 mg/L, about 25 to about 500 mg/L, about 100 to about 1,500 mg/L, or about 200 to about 1,000 mg/L, at least about 1,000 mg/L, at least about 1,200 mg/L, at least about at least 1,400 mg/L, at least about 1,600 mg/L, at least about 1,800 mg/L, at least about 2,800 mg/L, or at least about 7,000 mg/L. In some aspects, the amount of an individual steviol glycoside can exceed 7,000 mg/L. The amount of a combination of steviol glycosides (e.g., RebA, RebB, RebD, or RebM) accumulated can be from about 1 mg/L to about 7,000 mg/L, e.g., about 200 to about 1,500, at least about 2,000 mg/L, at least about 3,000 mg/L, at least about 4,000 mg/L, at least about 5,000 mg/L, at least about 6,000 mg/L, or at least about 7,000 mg/L. In some aspects, the amount of a combination of steviol glycosides can exceed 7,000 mg/L. In general, longer culture times will lead to greater amounts of product. Thus, the recombinant microorganism can be cultured for from 1 day to 7 days, from 1 day to 5 days, from 3 days to 5 days, about 3 days, about 4 days, or about 5 days.
It will be appreciated that the various genes and modules discussed herein can be present in two or more recombinant microorganisms rather than a single microorganism. When a plurality of recombinant microorganisms is used, they can be grown in a mixed culture to produce steviol and/or steviol glycosides. For example, a first microorganism can comprise one or more biosynthesis genes for producing a steviol glycoside precursor, while a second microorganism comprises steviol glycoside biosynthesis genes. The product produced by the second, or final microorganism is then recovered. It will also be appreciated that in some embodiments, a recombinant microorganism is grown using nutrient sources other than a culture medium and utilizing a system other than a fermenter.
Alternatively, the two or more microorganisms each can be grown in a separate culture medium and the product of the first culture medium, e.g., steviol, can be introduced into second culture medium to be converted into a subsequent intermediate, or into an end product such as RebA. The product produced by the second, or final microorganism is then recovered. It will also be appreciated that in some embodiments, a recombinant microorganism is grown using nutrient sources other than a culture medium and utilizing a system other than a fermenter.
Steviol glycosides and compositions obtained by the methods disclosed herein can be used to make food products, dietary supplements and sweetener compositions. See, e.g., WO 2011/153378, WO 2013/022989, WO 2014/122227, and WO 2014/122328.
For example, substantially pure steviol or steviol glycoside such as RebM or RebD can be included in food products such as ice cream, carbonated beverages, fruit juices, yogurts, baked goods, chewing gums, hard and soft candies, and sauces. Substantially pure steviol or steviol glycoside can also be included in non-food products such as pharmaceutical products, medicinal products, dietary supplements and nutritional supplements. Substantially pure steviol or steviol glycosides may also be included in animal feed products for both the agriculture industry and the companion animal industry. Alternatively, a mixture of steviol and/or steviol glycosides can be made by culturing recombinant microorganisms separately, each producing a specific steviol or steviol glycoside, recovering the steviol or steviol glycoside in substantially pure form from each microorganism and then combining the compounds to obtain a mixture comprising each compound in the desired proportion. The recombinant microorganisms described herein permit more precise and consistent mixtures to be obtained compared to current Stevia products.
In another alternative, a substantially pure steviol or steviol glycoside can be incorporated into a food product along with other sweeteners, e.g. saccharin, dextrose, sucrose, fructose, erythritol, aspartame, sucralose, monatin, or acesulfame potassium. The weight ratio of steviol or steviol glycoside relative to other sweeteners can be varied as desired to achieve a satisfactory taste in the final food product. See, e.g., U.S. 2007/0128311. In some embodiments, the steviol or steviol glycoside may be provided with a flavor (e.g., citrus) as a flavor modulator.
Compositions produced by a recombinant microorganism described herein can be incorporated into food products. For example, a steviol glycoside composition produced by a recombinant microorganism can be incorporated into a food product in an amount ranging from about 20 mg steviol glycoside/kg food product to about 1800 mg steviol glycoside/kg food product on a dry weight basis, depending on the type of steviol glycoside and food product. For example, a steviol glycoside composition produced by a recombinant microorganism can be incorporated into a dessert, cold confectionary (e.g., ice cream), dairy product (e.g., yogurt), or beverage (e.g., a carbonated beverage) such that the food product has a maximum of 500 mg steviol glycoside/kg food on a dry weight basis. A steviol glycoside composition produced by a recombinant microorganism can be incorporated into a baked good (e.g., a biscuit) such that the food product has a maximum of 300 mg steviol glycoside/kg food on a dry weight basis. A steviol glycoside composition produced by a recombinant microorganism can be incorporated into a sauce (e.g., chocolate syrup) or vegetable product (e.g., pickles) such that the food product has a maximum of 1000 mg steviol glycoside/kg food on a dry weight basis. A steviol glycoside composition produced by a recombinant microorganism can be incorporated into bread such that the food product has a maximum of 160 mg steviol glycoside/kg food on a dry weight basis. A steviol glycoside composition produced by a recombinant microorganism, plant, or plant cell can be incorporated into a hard or soft candy such that the food product has a maximum of 1600 mg steviol glycoside/kg food on a dry weight basis. A steviol glycoside composition produced by a recombinant microorganism, plant, or plant cell can be incorporated into a processed fruit product (e.g., fruit juices, fruit filling, jams, and jellies) such that the food product has a maximum of 1000 mg steviol glycoside/kg food on a dry weight basis. In some embodiments, a steviol glycoside composition produced herein is a component of a pharmaceutical composition. See, e.g., Steviol Glycosides Chemical and Technical Assessment 69th JECFA, 2007, prepared by Harriet Wallin, Food Agric. Org.; EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS), “Scientific Opinion on the safety of steviol glycosides for the proposed uses as a food additive,” 2010, EFSA Journal 8(4):1537; U.S. Food and Drug Administration GRAS Notice 323; U.S. Food and Drug Administration GRAS Notice Notice 329; WO 2011/037959; WO 2010/146463; WO 2011/046423; and WO 2011/056834.
For example, such a steviol glycoside composition can have from 90-99 weight % RebA and an undetectable amount of stevia plant-derived contaminants, and be incorporated into a food product at from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis.
Such a steviol glycoside composition can be a RebB-enriched composition having greater than 3 weight % RebB and be incorporated into the food product such that the amount of RebB in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis. Typically, the RebB-enriched composition has an undetectable amount of stevia plant-derived contaminants.
Such a steviol glycoside composition can be a RebD-enriched composition having greater than 3 weight % RebD and be incorporated into the food product such that the amount of RebD in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis. Typically, the RebD-enriched composition has an undetectable amount of stevia plant-derived contaminants.
Such a steviol glycoside composition can be a RebE-enriched composition having greater than 3 weight % RebE and be incorporated into the food product such that the amount of RebE in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis. Typically, the RebE-enriched composition has an undetectable amount of stevia plant-derived contaminants.
Such a steviol glycoside composition can be a RebM-enriched composition having greater than 3 weight % RebM and be incorporated into the food product such that the amount of RebM in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis. Typically, the RebM-enriched composition has an undetectable amount of stevia plant-derived contaminants.
In some embodiments, a substantially pure steviol or steviol glycoside is incorporated into a tabletop sweetener or “cup-for-cup” product. Such products typically are diluted to the appropriate sweetness level with one or more bulking agents, e.g., maltodextrins, known to those skilled in the art. Steviol glycoside compositions enriched for RebA, RebB, RebD, RebE, or RebM, can be package in a sachet, for example, at from 10,000 to 30,000 mg steviol glycoside/kg product on a dry weight basis, for tabletop use. In some embodiments, a steviol glycoside produced in vitro, in vivo, or by whole cell bioconversion
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
The Examples that follow are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the invention.
LC-MS analyses were performed on Waters ACQUITY UPLC® (Waters Corporation) with a Waters ACQUITY UPLC® BEH C18 column (2.1×50 mm, 1.7 μm particles, 130 Å pore size) coupled to a Waters ACQUITY TQD triple quadropole mass spectrometer with electrospray ionization (ESI) in negative mode.
Compound separation for Method A was achieved by a gradient of the two mobile phases: A (water with 0.1% formic acid) and B (MeCN with 0.1% formic acid) by increasing from 20% to 50% B between 0.3 to 2.0 min, increasing to 100% B at 2.01 min, holding 100% B for 0.6 min, and re-equilibrating for 0.6 min.
Compound separation for Method B was achieved by a gradient of the two mobile phases A (water with 0.1% formic acid) and B (MeCN with 0.1% formic acid) by increasing from 60% to 100% B in 2.5 min, holding 100% B for 0.1 min and re-equilibrating for 0.3 min.
The flow rate was 0.6 mL/min, and the column temperature was 55° C. Steviol glycosides were monitored using SIM (Single Ion Monitoring) and quantified by comparing with authentic standards. See Table 1 for m/z trace and retention time values of steviol glycosides detected.
Colonies of E. coli strains constructed to express a UGT polypeptide were placed into sterile 96 deep well plates with 1 mL of NZCYM bacterial culture broth comprising ampicillin. The plate was sealed and samples were allowed to grow overnight at 37° C., shaking at 200 rpm. The following day (i.e., Day 2), 50 μL of each culture was transferred to a new sterile 96 deep well plate with 1 mL of NZCYM bacterial culture broth comprising ampicillin and polypeptide expression inducers. The plate was sealed and samples were incubated at 20° C., shaking at 200 rpm for ˜20 h. On Day 3, the plate was centrifuged at 4000 rpm for 10 min at 4° C. After decanting the supernatant, 50 μL of a buffer comprising Tris-HCl, MgCl2, CaCl2, and protease inhibitors was added to each well and cells were resuspended by shaking at 200 rpm for 5 min at 4° C. The contents of each well (i.e., cell slurries) were then transferred to a PCR plate and sealed before freezing at −80° C. overnight. Frozen cell slurries were thawed at room temperature for up to 30 min. If the thawing mix was not viscous due to cell lysing, samples were frozen and thawed again. When samples were nearly thawed, 25 μL of binding buffer comprising DNase and MgCl2 was added to each well. The PCR plate was incubated at room temperature for 5 min, shaking at 500 rpm, until samples became less viscous. Finally, samples were centrifuged at 4000 rpm for 5 min, after which the supernatants were used to measure UGT activity, as described in Example 3.
UGT polypeptide samples prepared according to Example 2 were screened in vitro for activity on substrates including RebA, RebB, rubusoside, steviol, ent-kaurenoic acid, and 13-SMG by preparing a reaction mixture according to Table 2.
The reaction mixture was incubated overnight at 30° C. The reaction was stopped by adding 30 μL of 100% DMSO. The resultant mixture was diluted further with 90 μL 50% DMSO for LC-MS analysis according to Example 1. Both the products formed and the area-under-the-curve (AUC) values of each product are shown in Tables 3-7, organized by substrate.
As shown in Tables 3-7, 19-O-glycosylation, 13-O-glycosylation, and glycosyl-group glycosylation activity by UGT polypeptides on several substrates was observed, resulting in the formation of glycosides of ent-kaurenoic acid and steviol.
As shown in Table 8, UDPG1 (SEQ ID NO:185) and UGT75D1 (SEQ ID NO:205) produce relatively more rubusoside from 13-SMG than ent-kaurenoic acid+1Glc (#58) from ent-kaurenoic acid in vitro, compared to UGT74G1 (SEQ ID NO:4)
SA Gtase (SEQ ID NO:182, SEQ ID NO:183) was expressed with a p416-GPD vector in a steviol glycoside-producing S. cerevisiae strain comprising one or more copies of a recombinant gene encoding a GGPPS polypeptide (SEQ ID NO:19, SEQ ID NO:20), a recombinant gene encoding a truncated CDPS polypeptide (SEQ ID NO:39, SEQ ID NO:40), a recombinant gene encoding an KS polypeptide (SEQ ID NO:51, SEQ ID NO:52), a recombinant gene encoding a KO polypeptide (SEQ ID NO:59, SEQ ID NO:60), a recombinant gene encoding an ATR2 polypeptide (SEQ ID NO:91, SEQ ID NO:92), a recombinant gene encoding an EUGT11 polypeptide (SEQ ID NO:14/SEQ ID NO:15, SEQ ID NO:16), a recombinant gene encoding an KAH polypeptide (SEQ ID NO:93, SEQ ID NO:94), a recombinant gene encoding a CPR8 polypeptide (SEQ ID NO:85, SEQ ID NO:86), a recombinant gene encoding an UGT85C2 polypeptide (SEQ ID NO:5/SEQ ID NO:6/SEQ ID NO:149, SEQ ID NO:7) or a UGT85C2 variant (or functional homolog) of SEQ ID NO:7, a recombinant gene encoding a UGT74G1 polypeptide (SEQ ID NO:3, SEQ ID NO:4) of a UGT74G1 variant (or functional homolog) of SEQ ID NO:4, a recombinant gene encoding a UGT76G1 polypeptide (SEQ ID NO:8, SEQ ID NO:9) or a UGT76G1 variant (or functional homolog) of SEQ ID NO:9, and a recombinant gene encoding a UGT91D2e polypeptide (SEQ ID NO:10, SEQ ID NO:11) and a UGT91D2e variant (or functional homolog) of SEQ ID NO:11 such as a UGT91D2e-b (SEQ ID NO:12, SEQ ID NO:13).
The strain was incubated in 1 mL synthetic complete (SC) uracil dropout media at 30° C. for five days, shaking at 400 rpm. 50 μL of each culture was transferred into 50 μL DMSO, incubated at 80° C. for 10 min, and centrifuged at 3220 g for 5 min. 15 μL of the resulting supernatant was then transferred to 105 μL 50% DMSO for LC-MS analysis, which was carried out according to Example 1. Normalized area-under-the-curve (AUC) values for LC-MS derived peaks corresponding to RebD and RebM were about 0.25 μM/OD600 and 1.15 μM/OD600, respectively. Ent-kaurenoic acid+2Glc (#7), ent-kaurenoic acid+3Glc (isomer 1), and ent-kaurenoic acid+3Glc (isomer 2) accumulated at levels of about 200 AUC/OD600, 15 AUC/OD600, and 1000 AUC/OD600, respectively. 13-SMG, RebA, and Reb B accumulated at levels of about 4.8 μM/OD600, 2.5 μM/OD600, and 0.25 μM/OD600, respectively. Steviol+4Glc (#26), steviol+6Glc (isomer 1), steviol+7Glc (isomer 2), and kaurenol+3Glc (isomer 1 and/or 2) accumulated at levels of about 200 AUC/OD600, 15 AUC/OD600, 75 AUC/OD600, and 750 AUC/OD600, respectively.
Having described the invention in detail and by reference to specific embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. More specifically, although some aspects of the present invention are identified herein as particularly advantageous, it is contemplated that the present invention is not necessarily limited to these particular aspects of the invention.
S. rebaudiana
S. rebaudiana
S. rebaudiana
S. rebaudiana
S. rebaudiana
Oryza sativa
Oryza sativa
Stevia rebaudiana
Stevia rebaudiana
Gibberella fujikuroi
Mus musculus
Thalassiosira pseudonana
Streptomyces clavuligerus
Sulfolobus acidocaldarius
Synechococcus sp.
Stevia rebaudiana
Streptomyces clavuligerus
Bradyrhizobium japonicum
Zea mays
Arabidopsis thaliana
Stevia rebaudiana
Stevia rebaudiana
Zea mays
Populus trichocarpa
Arabidopsis thaliana
Phomopsis amygdali
Physcomitrella patens
Gibberella fujikuroi
Stevia rebaudiana
Lactuca sativa
Rubus suavissimus
Castanea mollissima
Thellungiella halophila
Vitis vinifera
Gibberella fujikuroi
Trametes versicolor
Arabidopsis thaliana
Stevia rebaudiana
Siraitia grosvenorii
Siraitia grosvenorii
Gibberella fujikuroi
Stevia rebaudiana
Stevia rebaudiana
Stevia rebaudiana
Rubus suavissimus
Arabidopsis thaliana
Arabidopsis thaliana
S. rebaudiana
Rubus suavissimus
Rubus suavissimus
Prunus avium
Prunus avium
Prunus mume
Prunus mume
Prunus mume
Prunus persica
Stevia rebaudiana
Stevia rebaudiana
Arabidopsis thaliana
Vitis vinifera
Medicago truncatula
Arabidopsis thaliana
Rubus suavissimus
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Stevia rebaudiana
Stevia rebaudiana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Gardenia jasminoides
Gardenia jasminoides
Arabidopsis thaliana
Arabidopsis thaliana
Catharanthus roseus
Catharanthus roseus
Arabidopsis thaliana
Arabidopsis thaliana
Streptomyces antibioticus
Streptomyces antibioticus
Oryza sativa
Oryza sativa
Nicotiana tabacum
Nicotiana tabacum
Siraitia grosvenorii
Siraitia grosvenorii
Crocus sativus
Crocus sativus
Crocus sativus
Crocus sativus
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Catharanthus roseus
Catharanthus roseus
Solanum lycopersicum
Solanum lycopersicum
Stevia rebaudiana
| Number | Date | Country | |
|---|---|---|---|
| 62337213 | May 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16098305 | Nov 2018 | US |
| Child | 17517818 | US |
